Methods and compositions for producing aluminum tolerant alfalfa

ABSTRACT

The invention relates to alfalfa plants and lines having aluminum tolerance. The invention also relates to parts of alfalfa plants from lines having aluminum tolerance, including seeds capable of growing aluminum tolerant alfalfa plants. Methods for the use and breeding of aluminum tolerant alfalfa plants are also provided.

INCORPORATION OF SEQUENCE LISTING

A computer readable form of the sequence listing is contained in thefile named “NBLE077_ST25.txt” which is 103 kb (measured in MS-Windows)and was created on Dec. 2, 2013, which is filed herewith and hereinincorporated by reference.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority of U.S. ProvisionalApplication Ser. No. 61/433,205, filed on Jan. 15, 2011, the disclosureof which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The invention relates to methods and compositions for producing alfalfaplants that tolerate the presence of aluminum in soil.

BACKGROUND OF THE INVENTION

Alfalfa (Medicago sativa subsp. sativa) is the most important foragelegume in the United States. Alfalfa is tetraploid, having 4homoeologous chromosomes for each of the 8 different chromosomes, for atotal of 32 chromosomes. It is highly desirable for hay production andpasture for livestock because it produces more protein per hectare thangrain or oilseed crops, and requires little or no nitrogen fertilizerbecause of its ability to carry out symbiotic nitrogen fixation.However, alfalfa is very sensitive to aluminum toxicity.

Aluminum (“Al”) toxicity causes similar symptoms in many plant species.Micromolar concentrations of Al⁺³ can damage the root system, sometimeswithin minutes of exposure. Damage to the root system then significantlyreduces yields due to an insufficient intake of water and othernutrients. Heavy applications of limestone and P fertilizer are commonlyused to prevent yield loss, but these amendments are often noteconomical or practical.

SUMMARY OF THE INVENTION

In a first aspect, there is provided a method for producing an aluminumtolerant alfalfa line or increasing the aluminum tolerance of an alfalfaline comprising introgres sing at least one chromosomal locuscontributing to aluminum tolerance from a parent alfalfa plant into analfalfa line. For example, the parent alfalfa plant may be an aluminumtolerant alfalfa plant, such as a plant that displays a reduction in oneor more symptoms of aluminum toxicity relative to a control plant whenthe plant is in contact with aluminum (e.g., a 10%, 25%, 50%, 75%, or90% reduction). Symptoms of aluminum toxicity that may be reduced inresistant plants include, but are not limited to, reduction orinhibition of root growth, increase in susceptibility to drought,nutrient deficiency, decreased yield, and leaf chlorosis and/ornecrosis. In certain embodiments, the chromosomal locus maps betweenloci MTIC95-146A and BG285-309A, MtBA36F01F1-126A and BG181-164A,BF228-153 and AL81-228, or MTIC84-18793 and BG234-251A on linkage group1; between loci RCS5744-229B33 and 1e04.tatc.4-1-232A or MTIC124-179Band MTIC169-113 on linkage group 3; between loci MTIC94-13538B andMstir12038-216, AW347-324A and BF184-28404, 1a09ggt5-1-252B andBG171-192, Mstri10127-123 and BF71-229, Mstri11701-17389 and1g05tct12-1-277, RCS4310-272B and MTIC332-1485860, or AL84-156A64A andMstir11989-111 on linkage group 4; between loci TC105099-111 and2c12.gga.5-1-165A, TC106861 and Mstri10743-120, Mstir10801-446A andMstri10743-120, or AW389-482 and BG157-154 on linkage group 5; betweenloci BI98-15458A and 8E92-199 or MTIC250-133 and 3d03.atc.5-1-239B244 onlinkage group 6; or between loci BG119-270 and MTIC183-170B orAW212-245A65 and BF26-289A96A04A on linkage group 7 (see e.g., markermaps provided in FIG. 5A-I). For example, the chromosomal locus may belinked to any of the markers in the regions between MTIC95-146A andBG285-309A, MtBA36F01F1-126A and BG181-164A, BF228-153 and AL81-228, orMTIC84-18793 and BG234-251A on chromosome 1; between RCS5744-229B33 and1e04.tatc.4-1-232A or MTIC124-179B and MTIC169-113 on chromosome 3;between MTIC94-13538B and Mstir12038-216, AW347-324A and BF184-28404,1a09ggt5-1-252B and BG171-192, Mstri10127-123 and BF71-229,Mstri11701-17389 and 1g05tct12-1-277, RCS4310-272B and MTIC332-1485860,or AL84-156A64A and Mstir11989-111 on chromosome 4; between TC105099-111and 2c12.gga.5-1-165A, TC106861 and Mstri10743-120, Mstir10801-446A andMstri10743-120, or AW389-482 and BG157-154 on chromosome 5; betweenBI98-15458A and 8E92-199 or MTIC250-133 and 3d03.atc.5-1-239B244 onchromosome 6; or between loci BG119-270 and MTIC183-170B or AW212-245A65and BF26-289A96A04A on chromosome 7 as provided in the maps of FIG.5A-I.

In some aspects, a method according to the invention comprises: (a)crossing a plant within the Medicago genus having aluminum tolerancewith a Medicago sativa plant lacking substantial aluminum tolerance toform a first population; (b) selecting one or more members of saidpopulation having aluminum tolerance; and (c) backcrossing progenyobtained to plants of a Medicago sativa variety otherwise lacking thealuminum tolerance to obtain an introgressed variety comprising aluminumtolerance. In certain embodiments, steps (b) and (c) may be repeateduntil an aluminum tolerance trait has been introgressed into the geneticbackground of a plant line that initially lacked aluminum tolerance suchthat the introgressed plant comprises less than about 50%, 25%, 10%, 5%,or 1% genomic material from the initial aluminum tolerant plant. In someembodiments, the initial cross of step (a) further comprises usingembryo rescue to form said first population. In certain embodiments, thesteps are repeated about 1, 2, 3, 4, 5, 6, or more times.

In certain aspects, a less-aluminum-tolerant alfalfa line is anagronomically elite line. For example, the less-aluminum-tolerantalfalfa line may be a commercial Medicago sativa line, such as a linethat is used to produce alfalfa hay or silage. The less aluminumtolerant alfalfa line may be a hybrid or inbred line. In certainspecific embodiments, the less-aluminum-tolerant alfalfa line is anycommercial variety that is well known to one skilled in the art.

In some aspects a less-aluminum-tolerant alfalfa parent plant or linemay contribute loci that enhance aluminum tolerance in progeny lines.For example, in some cases, the less-aluminum-tolerant alfalfa parentplant is a Medicago sativa NECS-141 plant. Loci contributing to aluminumtolerance that may be introgressed from such a parent plant include, butare not limited to, chromosomal loci mapping between BF228-153 andAL81-228 on linkage group 1; between 1a09ggt5-1-252B and BG171-192,Mstri10127-123 and BF71-229, Mstri11701-17389 and 1g05tct12-1-277, orRCS4310-272B and MTIC332-1485860 on linkage group 4; or betweenMTIC250-133 and 3d03.atc.5-1-239B244 on linkage group 6.

In some aspects, a more-aluminum-tolerant plant is another member of theMedicago genus, other than Medicago sativa L., such as Medicagotruncatula or Medicago trifolium. The plant may be a wild plant, or ahybrid or inbred line. In certain embodiments, themore-aluminum-tolerant alfalfa plant is Medicago sativa ssp. caeruleaaccession PI464724-25. In certain other embodiments themore-aluminum-tolerant alfalfa plant is a plant other than Medicagosativa ssp. caerulea accession PI464724-25. Loci contributing toaluminum tolerance that may be introgressed from a more aluminumtolerant parent plant include, but are not limited to, chromosomal locimapping between MTIC95-146A and BG285-309A, MtBA36F01F1-126A andBG181-164A, or MTIC84-18793 and BG234-251A on linkage group 1; betweenRCS5744-229B33 and 1e04.tatc.4-1-232A or MTIC124-179B and MTIC169-113 onlinkage group 3; between MTIC94-13538B and Mstir12038-216, AW347-324Aand BF184-28404, or AL84-156A64A and Mstir11989-111 on linkage group 4;between TC105099-111 and 2c12.gga.5-1-165A, TC106861 and Mstri10743-120,Mstir10801-446A and Mstri10743-120, or AW389-482 and BG157-154 onlinkage group 5; between B198-15458A and 8E92-199 on linkage group 6; orbetween BG119-270 and MTIC183-170B or AW212-245A65 and BF26-289A96A04Aon linkage group 7.

In a further aspect, there is provided a method for introgressingaluminum tolerance into an alfalfa line by marker-assisted selectionusing a marker linked to a chromosomal locus that contributes toaluminum tolerance in an alfalfa plant. In certain embodiments, themarker may be a marker that detects chromosomal insertions, deletions orother polymorphisms, such as simple sequence repeats and singlenucleotide polymorphisms (SNPs). In certain embodiments, a marker foruse according to the invention is between markers MTIC95-146A andBG285-309A, MtBA36F01F1-126A and BG181-164A, BF228-153 and AL81-228, orMTIC84-18793 and BG234-251A on linkage group 1; between RCS5744-229B33and 1e04.tatc.4-1-232A or MTIC124-179B and MTIC169-113 on linkage group3; between MTIC94-13538B and Mstir12038-216, AW347-324A and BF184-28404,1a09ggt5-1-252B and BG171-192, Mstri10127-123 and BF71-229,Mstri11701-17389 and 1g05tct12-1-277, RCS4310-272B and MTIC332-1485860,or AL84-156A64A and Mstir11989-111 on linkage group 4; betweenTC105099-111 and 2c12.gga.5-1-165A, TC106861 and Mstri10743-120,Mstir10801-446A and Mstri10743-120, or AW389-482 and BG157-154 onlinkage group 5; between BI98-15458A and 8E92-199 or MTIC250-133 and3d03.atc.5-1-239B244 on linkage group 6; or between BG119-270 andMTIC183-170B or AW212-245A65 and BF26-289A96A04A on linkage group 7. Forexample, the marker may be one of the markers detectable by one of theprimer pairs provided in Table 1 or Table 5 (SEQ ID NOs:1-560).

In still a further aspect, there is provided an alfalfa line produced bymethods according to the invention, wherein the line comprises aluminumtolerance and is agronomically elite. Progeny of such plants comprisingaluminum tolerance and an agronomically elite phenotype are alsoincluded as part of the invention.

In yet a further aspect, the invention provides an alfalfa plantcomprising aluminum tolerance wherein the plant is agronomically elite.For example, the alfalfa plant may be an inbred or hybrid plant. Atolerant alfalfa plant may display a reduction in one or more symptom ofaluminum toxicity. Symptoms that may be reduced in a tolerant plantinclude, but are not limited to, reduction or inhibition of root growth,increase in susceptibility to drought, nutrient deficiency, decreasedyield, and leaf chlorosis and/or necrosis. Progeny of such plantscomprising aluminum tolerance and an agronomically elite phenotype arealso included as part of the invention. Likewise, seeds of plantsaccording to the invention are also provided wherein the seeds produceagronomically elite plants comprising aluminum tolerance. Transgenicalfalfa plants are also provided as part of the instant invention. Incertain embodiments, the invention provides parts of a plant accordingto the invention. Plant parts included but are not limited to a leaf, anovule, pollen or a cell.

Plants according to the invention may be homozygous or heterozygous fora chromosomal locus linked to an aluminum tolerance phenotype. Infurther embodiments, the invention provides a seed of a plant accordingto the invention wherein the seed comprises a chromosomal locus linkedto aluminum tolerance.

In still a further aspect, an alfalfa plant according to the instantinvention comprises at least one additional trait of agronomic interest.

In yet another aspect, a tissue culture of regenerable cells of analfalfa plant according to the invention is provided. The tissue culturemay be capable of regenerating alfalfa plants capable of expressing allof the physiological and morphological characteristics of the startingplant (e.g., aluminum tolerance), and of regenerating plants havingsubstantially the same genotype as the starting plant. The regenerablecells in such tissue cultures may be derived, for example, from embryos,meristems, cotyledons, pollen, leaves, anthers, roots, root tips,pistil, flower, seed, or stalks. In still further embodiments, theinvention provides alfalfa plants regenerated from a tissue culture ofthe invention wherein the plants comprise aluminum tolerance.

In a further aspect, the present invention provides a method ofproducing progeny of a plant according to the invention, the methodcomprising the steps of: (a) preparing a progeny plant derived from analuminum tolerant plant, wherein said preparing comprises crossing aplant according to the invention with a second plant; and (b) crossingthe progeny plant with itself or a second plant to produce a seed of aprogeny plant of a subsequent generation. In further embodiments, themethod may additionally comprise: (c) growing a progeny plant of asubsequent generation from said seed of a progeny plant of a subsequentgeneration and crossing the progeny plant of a subsequent generationwith itself or a second plant; and repeating the steps for an additional3-10 generations to produce further progeny plants. The derived plantmay be an inbred line, and the aforementioned repeated crossing stepsmay be defined as comprising sufficient inbreeding to produce the inbredline. In the method, it may be desirable to select particular plantsresulting from step (c) for continued crossing according to steps (b)and (c). By selecting plants having one or more desirable traits, analuminum tolerant plant is obtained which possesses some of thedesirable traits of the line/hybrid as well as potentially otherselected traits.

In still a further aspect there is provided a method of vegetativelypropagating an alfalfa plant according to the invention comprising thesteps of: (a) collecting tissue capable of being propagated from a plantaccording to the invention; (b) cultivating said tissue to obtainproliferated shoots; (c) rooting said proliferated shoots to obtainrooted plantlets; and, optionally, (d) growing plants from the rootedplantlets.

In certain aspects, the present invention provides a method of producingfood or feed comprising: (a) obtaining a plant according to theinvention, wherein the plant has been cultivated to maturity, and (b)collecting plant tissue from the plant. Plants according to theinvention comprise, in certain aspects, a commercial alfalfa varietycomprising aluminum tolerance. Accordingly, alfalfa produced from suchplants may be of any variety.

In further aspects, the invention provides a method of making acommercial product comprising obtaining alfalfa according the inventionand producing a commercial product therefrom.

Embodiments discussed in the context of methods and/or compositions ofthe invention may be employed with respect to any other method orcomposition described herein. Thus, an embodiment pertaining to onemethod or composition may be applied to other methods and compositionsof the invention as well.

As used herein the terms “encode” or “encoding” with reference to anucleic acid are used to make the invention readily understandable bythe skilled artisan, however these terms may be used interchangeablywith “comprise” or “comprising” respectively.

As used herein the specification, “a” or “an” may mean one or more. Asused herein in the claim(s), when used in conjunction with the word“comprising”, the words “a” or “an” may mean one or more than one.

The use of the term “or” in the claims is used to mean “and/or” unlessexplicitly indicated to refer to alternatives only or the alternativesare mutually exclusive, although the disclosure supports a definitionthat refers to only alternatives and “and/or.” As used herein “another”may mean at least a second or more.

Throughout this application, the term “about” is used to indicate that avalue includes the inherent variation of error for the device, themethod being employed to determine the value, or the variation thatexists among the study subjects.

Other objects, features, and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1: Callus relative growth ratio (callus growth in medium withaluminum/callus growth in medium without aluminum) of six genotypesgrown in Blaydes callus induction medium with and without aluminum.

FIG. 2: Distribution of an aluminum-tolerant phenotype of theNECS141Altet4 mapping population. The phenotype graphed is the relativecallus growth ratio at 8 weeks of growth in the callus bioassay.

FIG. 3: Phenotypes of three genotypes of tetraploid alfalfa after 18days of growth in the whole-plant culture media assay.

FIG. 4: Frequency distribution of Al tolerance based on relative rootgrowth from the NECSAltet4 population from the whole plant assay inmedia.

FIG. 5A-I: Consensus linkage map and QTLs from Altet-4 chromosomes 1, 3to 7, and NECS-141 chromosomes 1, 4, and 6. Linkage map sectionsrepresent QTL regions identified from the callus bio-assay, whole plantassay in media, and soil-based assay (as indicated) identified frominterval mapping. QTL likelihood plots based on significant LOD scoresare shown for the following phenotypic assays: Callus-AVG=average ofcallus growth ratio; Al50=Relative root length in whole plant assay inmedia (pH7Al−/pH4Al+), with Al+ 50 μM; Al1K=Relative root length inwhole plant assay in media (pH7Al−/pH4Al+) with Al+ 1 mM); Rdmr=Relativeroot biomass in soil-based assay (experiment 1=Rdmr1, and experiment2=Rdmr2). X-axis=LOD score, Y axis, position in cM.

FIG. 6A-D: FIG. 6A, Altet-4: Mean callus growth ratio of QTL genotypeson chromosome 3 (74 cM) from callus bioassay. FIG. 6B, Altet-4: Meanrelative dry matter of roots of QTL genotypes on chromosome 4 (4 cM)from soil-based evaluations (Experiment 2, un-limed/limed). FIG. 6C,Altet-4: Mean relative dry matter of roots of QTL genotypes onchromosome 4 (38 cM) from soil-based evaluations (Experiment 1,un-limed/limed). FIG. 6D, Altet-4: Mean relative root growth (pH4Al+ 50μM/pH7Al−) of QTL genotypes on chromosome 7 (70 cM) from whole plantassay in media.

FIG. 7A-C: FIG. 7A, NECS-141: Mean relative root growth (pH4Al+ 1 mM/pH7Al−) of QTL genotypes on chromosome 1 (98 cM) from whole plant assay inmedia. FIG. 7B, NECS-141: Mean relative root growth (pH4Al+ 50 μM/pH7Al−) of QTL genotypes on chromosome 1 (98 cM) from whole plant assay inmedia. FIG. 7C, NECS-141: Mean relative dry matter of roots from QTLgenotypes on chromosome 4 (32 cM) based on soil-based evaluations(Experiment 2, un-limed/limed).

FIG. 8: Genetic linkage maps of Altet-4 and NECS-141. Consensus linkagemaps (left) and the four homologous linkage groups (H1-H4) (right) areshown for Chromosomes 1 through 8.

FIG. 9A-B: Al tolerance QTL on LG 1 explained 20.8% of the phenotypicvariation for total callus weight ratio (TCWR) from the callusbio-assay. A) Composite map of LG 1 from Altet-4 and QTL likelihoodplot. B) QTL allele effects at a given loci are based on the mean TCWRscore of six possible allelic combinations. Error bars represent anaverage of the standard errors of all genotypes within each alleliccombination. White numbers above the QTL genotype indicate the number ofindividuals with each allelic combination.

FIG. 10A-B: Al tolerance QTL on LG 4 of Altet-4 explained 15.2% of thephenotypic variation for total root length ratio (TRLR) in the wholeplant assay in media. A) Composite map of LG 4 from Altet-4 and QTLlikelihood plot. B) QTL allele effects at a given loci are based on themean TRLR score of six possible allelic combinations. Error barsrepresent an average of the standard errors of all genotypes within eachallelic combination. White numbers above the QTL genotype indicate thenumber of individuals with each allelic combination.

FIG. 11A-B: Al tolerance QTL on LG 7 of NECS-141 explained 21.7% of thephenotypic variation for total root length ratio (TRLR) from the wholeplant assay in media. A) Composite map of LG 7 from NECS-141 and QTLlikelihood plot. B) QTL allele effects at a given loci are based on themean TRLR score of six possible allelic combinations. Error barsrepresent an average of the standard errors of all genotypes within eachallelic combination. White numbers above the QTL genotype indicate thenumber of individuals with each allelic combination.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides alfalfa exhibiting tolerance or enhancedtolerance to aluminum. Such plants can be referred to as aluminumtolerant alfalfa varieties. Methods of producing aluminum tolerantalfalfa plants are also provided. Also disclosed herein are methods ofuse and derivatives of the aluminum tolerant alfalfa plants. Thesefindings can be used to enable implementation of effective molecularbreeding strategies through SNP genotyping and other high-throughputplatforms to accelerate the development of alfalfa cultivars withdesirable agronomic characteristics that are adapted to a range ofgrowing conditions, and are productive in acid and Al toxic soils.

The aluminum tolerant Medicago sativa alfalfa plants of the inventionmay bear one or more alleles conferring aluminum tolerance that havebeen introduced from other members of the Medicago genus employingtechniques described herein. According to the invention, such traits maybe introduced, for the first time, into agronomically elite varieties.Likewise, loci that contribute to aluminum tolerance have beenidentified in less-aluminum-tolerant plants. These loci can beintrogressed or maintained in a line to enhance aluminum tolerance.Aluminum tolerant alfalfa plants of the present invention may thusdisplay vigorous growth and other desirable properties for cultivation.

The invention also provides methods for introgression of aluminumtolerance into an alfalfa line. Through multiple rounds of backcrossing,chromosomal loci linked to aluminum tolerance may be introgressed intoany other genotype according to the invention. This allows production ofagronomically elite plants with aluminum tolerance. The backcrossingallows recovery of a starting genotype together with the desiredaluminum tolerance alleles. For example, aluminum tolerant lines maycomprise a genome that is 80%, 85%, 90%, 95%, 98%, or more Medicagosativa L. sequence from any particular background. Aluminum tolerantplants according to the invention may be defined, in certainembodiments, as “locus converted plants,” wherein essentially all of thedesired morphological and physiological characteristics of a variety arerecovered in addition to the characteristics of the single locustransferred into the variety via a backcrossing or by genetictransformation. Such lines may be heterozygous for chromosomal locilinked to aluminum tolerance or may be homozygous for such loci.Homozygous lines may have particular use, for example, as parents forselfing to produce inbred seed or crossing with a second elite alfalfaline for generating hybrid alfalfa seed.

Introgression of aluminum tolerance in accordance with the invention maybe affected by marker-assisted selection. In particular, the inventionprovides genetic markers genetically linked to alleles conferringaluminum tolerance. Thus, tracking of markers linked to these lociallows efficient identification of progeny plants harboring aluminumtolerance. High-throughput breeding techniques using marker-assistedselection can be used to rapidly introgress loci into an agronomicallyelite background and thereby produce commercially viable aluminumtolerant lines. Introgression of aluminum tolerance in alfalfa may alsobe performed by genomic selection. Genomic selection (GS) predicts thebreeding values of lines in a population by analyzing their phenotypesand marker scores (Heffner et al., Crop Sci. 49:1-12, 2009). GSincorporates all marker information in the prediction model, thusavoiding biased estimates of the marker effect and capturing more of thevariation due to small effect quantitative trait loci (QTL).

As used herein, an “agronomically elite” alfalfa plant or line refers toplants or varieties exhibiting traits appropriate for commercialproduction, which are well known to those of skill in the art. Forexample, agronomically elite plants are capable of producing acommercial scale hay or silage yield. In certain aspects, agronomicallyelite plants and lines produce alfalfa of uniform size, color andquality. Agronomically elite lines may also exhibit desirable hardinesstraits, such as disease resistance, cold tolerance, environmental stresstolerance, persistence, forage quality, and nutrient utilization, or usetraits such as improved harvestability.

As used herein, a “control alfalfa plant” is any alfalfa plantsusceptible to aluminum (aluminum susceptible), including typicalcommercially available and wild relatives of modern alfalfa plants. Acontrol alfalfa plant is also grown under similar environmentalconditions to a test plant according to the present disclosure.

As used herein, a “hybrid alfalfa plant” includes a plant resultingdirectly or indirectly from crosses between populations, breeds orcultivars within the species Medicago sativa. This also refers to plantsresulting directly or indirectly from crosses between different specieswithin the Medicago genus (e.g., interspecific hybrids resulting fromcrosses between Medicago sativa and Medicago truncatula or crossesbetween Medicago sativa and Medicago trifolium).

As used herein an “aluminum tolerant alfalfa plant” displays anincreased tolerance to aluminum, or a decrease in the development ofsymptoms of aluminum susceptibility, when compared to the parentalMedicago sativa plant or a control alfalfa line grown under similarenvironmental conditions.

As used herein, a descendent or progeny of a particular plant includesnot only, without limitation, the products of any initial cross (be it abackcross or otherwise) between two plants, but all descendants whosepedigree traces back to the original cross. In an aspect of the presentinvention, the descendent contains about 50%, 25%, 12.5%, 6%, 3%, 1%, orless nuclear DNA from an aluminum tolerant alfalfa plant and expressesthat genetic material to provide at least a portion of the plant'saluminum tolerance.

Aluminum tolerant alfalfa plants also include alfalfa cultivars, linesor varieties having tolerance to aluminum, referred to herein asaluminum tolerant alfalfa cultivars, aluminum tolerant alfalfa lines, oraluminum tolerant alfalfa varieties respectively. Aluminum tolerantalfalfa cultivars, aluminum tolerant alfalfa lines, or aluminum tolerantalfalfa varieties may have been bred and selected for at least aluminumtolerance and may also have been selected for other desirable traits.

As used herein, a “female parent” refers to an alfalfa plant that is therecipient of pollen from a male donor line, which successfullypollinates an egg. A female parent can be any alfalfa plant that is therecipient of pollen. Such female parents can be male sterile, forexample, because of genetic male sterility, cytoplasmic male sterility,or because they have been subject to emasculation of the stamens.Genetic or cytoplasmic male sterility can be manifested in differentmanners, such as sterile pollen, malformed or stamenless flowers,positional sterility, and functional sterility.

As used herein, “cytoplasmic male sterility” refers to plants that arenot usually capable of breeding from self-pollination, but are capableof breeding from cross-pollination.

As used herein, “linkage” or “genetic linkage” is a phenomenon whereinalleles on the same chromosome tend to segregate together more oftenthan expected by chance if their transmission was independent.

As used herein, a “marker” is an indicator for the presence of at leastone phenotype, genotype, or polymorphism. Markers include, but are notlimited to, single nucleotide polymorphisms (SNPs), cleavable amplifiedpolymorphic sequences (CAPS), amplified fragment length polymorphisms(AFLPs), restriction fragment length polymorphisms (RFLPs), simplesequence repeats (SSRs), simple sequence length polymorphisms (SSLPs),insertion(s)/deletion(s) (INDEL(s)), and random amplified polymorphicDNA (RAPD) sequences. A marker may be codominant and completelyheritable (both alleles at a locus in a diploid heterozygote are readilydetectable), with no environmental variance component, i.e.,heritability of 1. A “nucleic acid marker” as used herein means anucleic acid molecule that is capable of being a marker for detecting apolymorphism, phenotype, or both associated with aluminum tolerance. A“molecular marker” as used herein means a nucleic acid molecule that iscapable of being a marker for detecting a polymorphism, phenotype, orboth associated with aluminum tolerance. Genetic maps and markers foruse in alfalfa are known in the art (Brummer et al., Theor Appl Genet86:329-332, 1993; Echt et al., Genome 37:61-711993; Kiss et al., Mol GenGenet 238:129-137, 1993; Brower et al., Crop Sci 40:1387-1396, 2000;Robins et al., Crop Sci 48:1780-1786, 2008; Robins et al., Crop Sci 471-10, 2007).

As used herein, a “desirable trait” or “desirable traits” that may beintroduced into aluminum tolerant alfalfa plants by breeding may bedirected to the alfalfa plant. Desirable alfalfa plant traits that maybe independently selected include, but are not limited to, plant vigor,leaf shape, leaf length, leaf color, plant height, time to maturity,adaptation to field growth, persistance, forage quality, and resistanceto one or more diseases or disease causing organisms. Any combination ofdesirable alfalfa traits may be combined with aluminum tolerance.

The present invention provides for one or more aluminum tolerant alfalfaplants. The aluminum tolerance of any alfalfa plant provided herein canbe a tolerance to high concentrations of aluminum or a tolerance to lowconcentrations of aluminum, wherein either the high or low concentrationof aluminum would cause symptoms in a non-aluminum-tolerant alfalfaplant. The aluminum tolerance of an alfalfa plant provided herein can bemeasured by any means available in the art.

In one aspect, the aluminum tolerance of an alfalfa plant is determinedusing a callus or tissue culture assay. The assay may comprise inducingcallus formation, transferring one part of the induced callus to agrowth medium comprising aluminum, and a second part of the callus to agrowth medium which does not comprise aluminum. The growth medium may beBlaydes callus induction medium, and the callus may be grown incontrolled growth chambers at 25° C. and with an 18-hour lightphotoperiod. The assay may further comprise weighing the callus. Theassay may further comprise comparing the relative weights or amount ofgrowth between the two parts of the callus.

In another aspect, the aluminum tolerance of an alfalfa plant isdetermined using a whole-plant culture media assay. The assay maycomprise growing vegetatively propagated alfalfa clones or stem cuttingsin culture media comprising 400 μM CaCl₂, 1.4% gel rite, 0 or 50 μM Al⁺³in the form of AlCl₃, and pH 7.0 or 4.0 adjusted using 1 N HCl, and thealfalfa may be grown in controlled-environment growth chambers at 25° C.with an 18-hour light photoperiod.

Root growth may be quantified using winRHIZO® software (RegentInstruments, Québec, Canada) to determine aluminum tolerance. Forexample, total root length, lateral root numbers, and branching may bequantified. The absolute root growth and ratio of root characteristics(biomass, length, and branching) after 3 weeks of growth in eitheraluminum-containing media or aluminum-free media may also be used fordetermining aluminum tolerance.

In another aspect, the alfalfa plants and lines provided hereindemonstrate little or no aluminum toxicity symptoms after treatment withaluminum. In some aspects, an aluminum tolerant alfalfa genotypedemonstrates aluminum toxicity symptoms in less than 10%, 9%, 8%, 7%,6%, 5%, 4%, 3% 2%, or 1% of alfalfa plants of that genotype.

Aluminum tolerant alfalfa plants may exhibit a delay in the onset ofaluminum toxicity symptoms relative to a non-tolerant control alfalfaplant. In some embodiments, the aluminum tolerant alfalfa plants exhibita delay of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more daysin the onset of aluminum toxicity symptoms relative to a control alfalfaplant. In other embodiments, the aluminum tolerant alfalfa plantsexhibit a delay of at least 7 or more days, 10 or more days, or 14 ormore days in the onset of aluminum toxicity symptoms relative to acontrol alfalfa plant.

In one aspect, the alfalfa plant is a seedling at the time of aluminumexposure. In some aspects, the alfalfa plant is a seedling at thetrifoliate leaf stage of development at the time of aluminum exposure.In one aspect, aluminum toxicity symptoms can be measured at any timeafter aluminum exposure of an alfalfa plant. In other aspects, symptomscan be measured 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, or more days after exposure. In another aspect, thealfalfa plant is any age of plant at the time of exposure.

In another aspect, the alfalfa plant is a callus at the time of aluminumexposure. In some aspects, the callus has been allowed to form for abouttwo weeks in Blaydes callus induction medium before exposure. In oneaspect, aluminum toxicity symptoms can be measured at any time afteraluminum exposure of an alfalfa callus. In other aspects, symptoms canbe measured 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, or more weeks after exposure. In another aspect, the alfalfacallus is any age of callus at the time of exposure.

In another aspect, the alfalfa plant is a vegetatively propagatedalfalfa clone or stem cutting at the time of aluminum exposure. In someaspects, the vegetatively propagated alfalfa clone or stem cutting hasbeen allowed to develop in medium comprising 400 μM CaCl₂, 1.4% gelrite, 0 or 50 μM Al⁺³ in the form of AlCl₃ before exposure. In oneaspect, aluminum toxicity symptoms can be measured at any time afteraluminum exposure of an alfalfa plant. In other aspects, symptoms can bemeasured 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, or more days after exposure. In another aspect, the alfalfaplant is any age of plant at the time of exposure.

Aluminum tolerant alfalfa plants of the present invention may exhibit anincrease in callus relative growth ratios after exposure to aluminumwhen compared to the relative growth rate of a control alfalfa callusexposed to aluminum. In one aspect, the aluminum tolerant alfalfa callusexhibit a 1%, 2%, 5%, 10%, 15%, 20%, or more increase in callus relativegrowth ratio relative to a control alfalfa plant after exposure toaluminum.

The present invention provides for a seed of an alfalfa plant capable ofproducing an aluminum tolerant alfalfa plant. In one aspect, thealuminum tolerant alfalfa plant can be an open-pollinated variety, ahybrid parent inbred line, or a male sterile line.

The aluminum tolerant alfalfa plants of the present invention can bealuminum tolerant alfalfa lines adapted for field alfalfa production orany other growing environment. In one aspect, the aluminum tolerantalfalfa plants of the present invention are adapted for open fieldalfalfa production.

The present invention also provides for an intra-specific hybrid alfalfaplant having aluminum tolerance developed from aluminum tolerant alfalfaplants. In another aspect, those intra-specific hybrid alfalfa plantsexhibit aluminum tolerance after exposure to aluminum.

Agronomically elite alfalfa plants appropriate for use in a commercialproduction field represent various aspects of the present invention. Inone aspect, certain alfalfa traits, including, for example, hay orsilage quality, may be important to the commercial value of the crop.

A further aspect of the invention relates to tissue cultures of thealuminum tolerant alfalfa plants described herein. As used herein, theterm “tissue culture” indicates a composition comprising isolated cellsof one or more types, or a collection of such cells organized into partsof a plant. Tissue culture includes, but is not limited to, compositionscomprising protoplasts and calli. Tissue culture also includes, but isnot limited to, compositions comprising plant cells that are present inintact plant tissues, or parts of plants, such as embryo, leaf,peduncle, pedicel, anther, meristem, tip and segments of root, stump andstem, explants, and the like. In one aspect, a tissue culture comprisesembryos, protoplasts, meristematic cells, pollen, leaves, anthers, orcells derived from immature tissues of these plant parts. Means forpreparing and maintaining plant tissue cultures are well known in theart. Examples of processes of tissue culturing and regeneration ofalfalfa are described in, for example, Parrot and Bouton, Crop Sci.,(1990) 30:387-389. In some aspects, tissue culture of the aluminumtolerant alfalfa plants described herein relates to the culture ofprotoplasts, calli, or plant cells, that are isolated from, or presentin, intact parts of the aluminum tolerant alfalfa plants describedherein. In another aspect, tissue culture refers to the culture ofprotoplasts, calli, or plant cells, that are isolated from, or presentin, intact parts of one or more aluminum tolerant plants selected fromthe group consisting of Altet1, Altet2, Altet3, and/or Altet4, andaluminum tolerant descendants thereof, including those produced bycrosses or backcrosses. In yet another aspect, tissue culture of thealuminum tolerant alfalfa plants described herein relates to the cultureof protoplasts, calli, or plant cells, that are isolated from, orpresent in, intact parts of the aluminum tolerant plants describedherein.

Once aluminum tolerant alfalfa plants are produced, the plantsthemselves can be cultivated in accordance with conventional procedures.Aluminum tolerant descendants of aluminum tolerant alfalfa plants may beobtained through sexual reproduction. The seeds resulting from sexualreproduction can be recovered from the aluminum tolerant alfalfa plantsand planted or otherwise grown as a means of propagation. Aluminumtolerant descendants may also be obtained from aluminum tolerant alfalfaplants through asexual reproduction. Protoplast or propagules (e.g.,cuttings, scions, or rootstocks) can be recovered from aluminum tolerantalfalfa plants, or parts thereof, and may be employed to propagatealuminum tolerant alfalfa plants.

The present invention also provides for and includes a container ofalfalfa seeds in which alfalfa plants grown from greater than 50% of theseeds have resistance or partial aluminum tolerance. In another aspect,alfalfa plants grown from greater than 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 98%, or 99% of the alfalfa seeds in the container havealuminum tolerance. Another aspect of the invention relates to seedsfrom an alfalfa plant selected from the group consisting of Altet1,Altet2, Altet3, Altet4, and aluminum tolerant descendents thereof,wherein alfalfa plants grown from about 50%, or greater than 50%, of theseeds have resistance or partial aluminum tolerance.

The container of alfalfa seeds can contain any number, weight or volumeof seeds. For example, a container can contain about, or greater thanabout, 10, 25, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000,1500, 2000, 2500, 3000, 3500, 4000, or more seeds. In another aspect, acontainer can contain about, or greater than about, 1 gram, 5, 10, 15,20, 25, 50, 100, 250, 500, or 1,000 grams of seeds. Alternatively, thecontainer can contain about or at least, or greater than, about 1 ounce,2, 3, 4, 5, 6, 7, 8, 9, 10 ounces, 1 pound, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12 pounds or more of seeds.

Containers of alfalfa seeds can be any container available in the art.For example, a container can be a box, a bag, a packet, a pouch, a taperoll, a foil, a pail, or a tube.

One aspect of the invention relates to dried, or otherwise processedalfalfa hay, produced by an alfalfa plant having a genome that comprisesat least one genetic locus giving rise to aluminum tolerance whenexpressed in an alfalfa plant. Processed alfalfa can be in the form of,but is not limited to, hay, silage, haylage, fermented hay, orgreenchop. In some aspects, the dried, or otherwise processed alfalfa,is from an alfalfa plant selected from one or more of the groupconsisting of Altet1, Altet2, Altet3 and/or Altet4, and aluminumtolerant descendents thereof.

The present invention includes and provides for Medicago sativa plantshaving at least one allele for an aluminum tolerance trait. The aluminumtolerant alfalfa plants can be either heterozygous or homozygous for thealuminum tolerance trait. In one embodiment, the aluminum tolerancetrait can be linked to variations in a single gene (e.g., linked to oneor more alleles of a single gene). In another embodiment, the aluminumtolerance trait can be linked to variations at one or one or morequantitative trait loci (QTL). In a yet another embodiment, the aluminumtolerant alfalfa plants are homozygous for the aluminum tolerance trait.In one aspect, the genetic loci derived from an aluminum tolerantalfalfa plant can be identified using genetic markers.

The present invention provides for an aluminum tolerant alfalfa planthaving less than or equal to 50% of its genome derived from a non-M.sativa aluminum tolerant plant that can be crossed directly, orindirectly (e.g., through tissue culture manipulation, or through theuse of a bridging species) with Medicago sativa. The present inventionalso provides for descendents of alfalfa plants having aluminumtolerance.

One aspect of the present invention provides for an aluminum tolerantalfalfa plant that contains a genetic marker or a complement to agenetic marker linked to one or more aluminum tolerance loci. Anotheraspect of the invention is an alfalfa plant that contains at least 1, 2,3, or 4 sequences complementary to markers linked to an aluminumtolerance locus. In another aspect, an alfalfa plant can containsequence complementary to any combination of markers linked to thealuminum tolerance locus.

As used herein linkage of two loci, including a marker sequence and anallele imparting a desired trait such as aluminum tolerance, may begenetic or physical or both. In one aspect of the invention, a nucleicacid marker and genetic locus conferring aluminum tolerance aregenetically linked and, for example, are located less than 50 cM fromone another. In particular embodiments, the marker and locus may exhibita LOD score of greater than 2.0, as judged by interval mapping for thealuminum tolerance trait based on maximum likelihood methods describedby Lander and Botstein, Genetics, 121:185-199 (1989), and implemented inthe software package MAPMAKER (default parameters). In otherembodiments, the marker and region conferring aluminum tolerance aregenetically linked and exhibit a LOD score of greater than 3.0, or a LODscore of greater than 3.5, or a LOD score of about 4.0 based uponinterval mapping.

In another aspect, the nucleic acid marker is genetically linked at adistance of between about 0 and about 49 centimorgans (cM) to thealuminum tolerance locus. In other embodiments, the distance between thenucleic acid marker and the aluminum tolerance locus is between about 0and about 30 cM, or between about 0 and about 20 cM, or between about 0and about 15 cM, or between about 0 and about 10 cM, or between about 0and about 5 cM, or less. See, for example, FIG. 5 which providedrelative distance in cM between identified loci.

In another aspect, the nucleic acid molecule may be physically linked toan aluminum tolerance locus. In some aspects, the nucleic acid markerspecifically hybridizes to a nucleic acid molecule having a sequencethat is within about 30 Mbp, or about 20 Mbp, or about 15 Mbp, or about10 Mbp, or about 5 Mbp of an aluminum tolerance locus.

As used herein, two nucleic acid molecules are said to be capable ofhybridizing to one another if the two molecules are capable of formingan anti-parallel, double-stranded nucleic acid structure. Conventionalstringency conditions are described by Sambrook et al., MolecularCloning, A Laboratory Manual, 2nd Ed., Cold Spring Harbor Press, ColdSpring Harbor, N.Y. (1989) and by Haymes et al., Nucleic AcidHybridization, A Practical Approach, IRL Press, Washington, D.C. (1985).Departures from complete complementarity are therefore permissible, aslong as such departures do not completely preclude the capacity of themolecules to form a double-stranded structure. Thus, in order for anucleic acid molecule to serve as a primer or probe it need only besufficiently complementary in sequence to be able to form a stabledouble-stranded structure under the particular solvent and saltconcentrations employed.

Appropriate stringency conditions which promote DNA hybridization, forexample, 6.0× sodium chloride/sodium citrate (SSC) at about 45° C.,followed by a wash of 2.0×SSC at 50° C., are known to those skilled inthe art or can be found in Current Protocols in Molecular Biology, JohnWiley & Sons, N.Y. (1989), 6.3.1-6.3.6. In some embodiments,hybridization conditions can be high, moderate or low stringencyconditions. High stringency conditions, for example, typically include awash step at 65° C. in 0.2×SSC.

The specificity of hybridization can be affected by post-hybridizationwashes. For example, the salt concentration in the wash step can beselected from a low stringency of about 2.0×SSC at 50° C. to a moderatestringency of about 1.0×SSC at 50° C. to a high stringency of about0.2×SSC at 50° C. In addition, the temperature in the wash step can beincreased from low stringency conditions at room temperature, about 22°C., to moderate stringency conditions at about 50° C., to highstringency conditions at about 65° C. Both temperature and saltconcentration may be varied, or either the temperature or the saltconcentration may be held constant while the other variable is changed.In some aspects, the wash step can be performed for 5, 10, 15, 20, 25,30, or more minutes. In another aspect, the wash step is performed forabout 20 minutes. In yet another aspect, the wash step can be repeated1, 2, 3, 4, or more times using the selected salt concentration,temperature, and time. In another aspect, the wash step is repeatedtwice.

A genetic marker profile of a plant may be predictive of the agronomictraits of a hybrid plant produced using that plant as a parent. Forexample, if an inbred plant having a known genetic marker profile andphenotype is crossed with a second inbred plant having a known geneticmarker profile and phenotype, it is possible to predict the phenotype ofthe F₁ hybrid based on the combined genetic marker profiles of theparent inbred plants. Methods for prediction of hybrid performance fromgenetic marker data are disclosed in U.S. Pat. No. 5,492,547, thedisclosure of which is specifically incorporated herein by reference inits entirety. Such predictions may be made using any suitable geneticmarker, for example, SSRs, INDELs, RFLPs, AFLPs, SNPs, or isozymes.

Additional markers, such as SSRs, AFLP markers, RFLP markers, RAPDmarkers, phenotypic markers, SNPs, isozyme markers, or microarraytranscription profiles that are genetically linked to or correlated withaluminum tolerance can be utilized (Walton, Seed World 22-29 (July,1993); Burow and Blake, Molecular Dissection of Complex Traits, 13-29,Eds. Paterson, CRC Press, New York (1988)). Methods to isolate suchmarkers are known in the art. For example, locus-specific SSRs can beobtained by screening an alfalfa genomic library for SSRs, sequencing of“positive” clones, designing primers which flank the repeats, andamplifying genomic DNA with these primers.

The genetic linkage of marker molecules to aluminum tolerance can beestablished by a gene mapping model such as, without limitation, theflanking marker model reported by Lander and Botstein, Genetics,121:185-199 (1989), and the interval mapping, based on maximumlikelihood methods described by Lander and Botstein, Genetics,121:185-199 (1989), and implemented in the software package MAPMAKER.

A maximum likelihood estimate (MLE) for the presence of a marker iscalculated, together with an MLE assuming no trait effect, to avoidfalse positives. A log₁₀ of an odds ratio (LOD) is then calculated as:LOD=log₁₀ (MLE for the presence of a trait (MLE given no linked trait)).

The LOD score essentially indicates how much more likely the data are tohave arisen assuming the presence of a resistance allele rather than inits absence. The LOD threshold value for avoiding a false positive witha given confidence, say 95%, depends on the number of markers and thelength of the genome. Graphs indicating LOD thresholds are set forth inLander and Botstein, Genetics, 121:185-199 (1989), and further describedby Ars and Moreno-Gonzalez, Plant Breeding, Hayward, Bosemark, Romagosa(eds.) Chapman & Hall, London, pp. 314-331 (1993).

Selection of appropriate mapping or segregation populations is importantin trait mapping. The choice of appropriate mapping population dependson the type of marker systems employed (Tanksley et al., Molecularmapping plant chromosomes. Chromosome structure and function: Impact ofnew concepts, J. P. Gustafson and R. Appels (eds.), Plenum Press, NewYork, pp. 157-173 (1988)). Consideration must be given to the source ofparents (adapted vs. exotic) used in the mapping population. Chromosomepairing and recombination rates can be severely disturbed (suppressed)in wide crosses (adapted×exotic) and generally yield greatly reducedlinkage distances. Wide crosses will usually provide segregatingpopulations with a relatively large array of polymorphisms when comparedto progeny in a narrow cross (adapted×adapted).

The present application provides a genetic complement of the alfalfalines described herein. Further provided is a hybrid genetic complement,wherein the complement is formed by the combination of a haploid geneticcomplement from elite inbred alfalfa lines described herein and anotherhaploid genetic complement. Means for determining such a geneticcomplement are well known in the art.

As used herein, the phrase “genetic complement” means an aggregate ofnucleotide sequences, the expression of which defines the phenotype of aplant, such as a Medicago sativa alfalfa plant or a cell or tissue ofthat plant. By way of example, a Medicago sativa alfalfa plant isgenotyped to determine a representative sample of the inherited markersit possesses. Markers may be inherited in codominant fashion so that thepresence of both alleles at a diploid or tetraploid locus is readilydetectable, and they are free of environmental variation, i.e., theirheritability is close to, or equal to, 1. This genotyping is may beperformed on at least one generation of the descendant plant for whichthe numerical value of the trait or traits of interest are alsodetermined. The array of single locus genotypes is expressed as aprofile of marker alleles, two at each locus for a diploid plant. Themarker allelic composition of each locus can be either homozygous orheterozygous. Homozygosity is a condition where both alleles at a locusare characterized by the same conditions of the genome at a locus (e.g.,the same nucleotide sequence). Heterozygosity refers to differentconditions of the genome at a locus. Potentially any type of geneticmarker could be used, for example, simple sequence repeats (SSRs),insertion/deletion polymorphism (INDEL), restriction fragment lengthpolymorphisms (RFLPs), amplified fragment length polymorphisms (AFLPs),single nucleotide polymorphisms (SNPs), and isozymes.

Considerable genetic information can be obtained from a completelyclassified F₂ population using a codominant marker system (Mather,Measurement of Linkage in Heredity: Methuen and Co., (1938)). An F₂population is the first generation of self or sib pollination after thehybrid seed is produced. Usually, a single F₁ plant is self or sibpollinated to generate a population segregating for the nuclear-encodedgenes in a Mendelian (1:2:1) fashion.

In contrast to the use of codominant markers, using dominant markersoften requires progeny tests (e.g., F₃ or back cross self families) toidentify heterozygous individuals in the preceding generation. Theinformation gathered can be equivalent to that obtained in a completelyclassified F₂ population. This procedure is, however, often prohibitivebecause of the cost and time involved in progeny testing. Progenytesting of F₂ individuals is often used in map construction where erroris associated with single-plant phenotyping, or when sampling the plantsfor genotyping affects the ability to perform accurate phenotyping, orwhere trait expression is controlled by a QTL. Segregation data fromprogeny test populations (e.g., F₃ or backcrossed or selfed families)can be used in trait mapping. Marker-assisted selection can then beapplied to subsequent progeny based on marker-trait map associations(F₂, F₃), where linkage has not been completely disassociated byrecombination events (i.e., linkage disequilibrium).

Recombinant inbred lines (RILs) (genetically related lines; usually >F₅)can be used as a mapping population. RILs can be developed by selfing F2plants, then selfing the resultant F3 plants, and repeating thisgenerational selfing process, thereby increasing homozygosity.Information obtained from dominant markers can be maximized by usingRILs because all loci are homozygous or nearly so. Under conditions oftight linkage (i.e., about <10% recombination), dominant and co-dominantmarkers evaluated in RIL populations provide more information perindividual than either marker type in backcross populations (Reiter etal., Proc. Natl. Acad. Sci. (U.S.A.) 89:1477-1481, 1992). However, asthe distance between markers becomes larger (i.e., loci become moreindependent), the information in RIL populations decreases dramaticallywhen compared to codominant markers.

Backcross populations can be utilized as mapping populations. Abackcross population (BC) can be created by crossing an F₁ to one of itsparents. Typically, backcross populations are created to recover thedesirable traits (which may include most of the genes) from one of therecurrent parental (the parent that is employed in the backcrosses)while adding one or a few traits from the second parental, which isoften referred to as the donor. A series of backcrosses to the recurrentparent can be made to recover most of the recurrent parent's desirabletraits. Thus a population is created consisting of individuals nearlylike the recurrent parent, wherein each individual carries varyingamounts or a mosaic of genomic regions from the donor parent. Backcrosspopulations can be useful for mapping dominant markers particularly ifall loci in the recurrent parent are homozygous and the donor andrecurrent parent have contrasting polymorphic marker alleles (Reiter etal., Proc. Natl. Acad. Sci. (U.S.A.) 89:1477-1481, 1992).

Information obtained from backcross populations using either codominantor dominant markers is less than that obtained from completelyclassified F₂ populations because recombination events involving one,rather than two, gametes are sampled per plant. Backcross populations,however, are more informative (at low marker saturation) when comparedto RILs as the distance between linked loci increases in RIL populations(i.e., about 15% recombination). Increased recombination can bebeneficial for resolution of tight linkages, but may be undesirable inthe construction of maps with low marker saturation.

Near-isogenic lines (NIL) created by many backcrosses to produce anarray of individuals that are nearly identical in genetic compositionexcept for the trait or genomic region under interrogation can be usedas a mapping population. In mapping with NILs, only a portion of theloci are polymorphic between the parentals are expected to segregate inthe highly homozygous NIL population. Those loci that are polymorphic ina NIL population, however, are likely to be linked to the trait ofinterest.

Bulk segregant analysis (BSA) is a method developed for the rapididentification of linkage between markers and traits of interest(Michelmore, et al., Proc. Natl. Acad. Sci. (U.S.A.) 88:9828-9832,1991). In BSA, two bulk DNA samples are drawn from a segregatingpopulation originating from a single cross. These bulk samples containindividuals that are identical for a particular trait (e.g., resistantor susceptible to a particular pathogen) or genomic region but arbitraryat unlinked regions (i.e., heterozygous). Regions unlinked to the targettrait will not differ between the bulked samples of many individuals inBSA.

In another aspect, the present invention provides a method of producingan aluminum tolerant alfalfa plant comprising: (a) crossing an aluminumtolerant alfalfa line with a second alfalfa line lacking aluminumtolerance to form a segregating population; (b) screening the populationfor aluminum tolerance; and (c) selecting one or more members of thepopulation having said aluminum tolerance. In one aspect, plants areidentified as aluminum tolerant prior to conducting one or more crosses.In one aspect, plants can be selected on the basis of partial orcomplete aluminum tolerance. In one aspect, the segregating populationis self-crossed and the subsequent population is screened forresistance.

In another aspect, the present invention provides a method ofintrogressing aluminum tolerance into an alfalfa plant comprising: (a)crossing at least a first aluminum tolerant alfalfa line with a secondalfalfa line to form a segregating population; (b) screening saidpopulation for aluminum tolerance; and (c) selecting at least one memberof said population exhibiting aluminum tolerance. In one aspect, plantsare identified as aluminum tolerant prior to conducting one or morecrosses. In one aspect, the segregating population is self-crossed andthe subsequent population is screened for resistance.

Aluminum tolerant alfalfa plants of the present invention can be partof, or generated from, a breeding program. The choice of breeding methoddepends on the mode of plant reproduction, the heritability of thetrait(s) being improved, and the type of cultivar used commercially(e.g., F₁ hybrid cultivar, pure line cultivar, etc). Selected,non-limiting approaches for breeding the plants of the present inventionare set forth below. A breeding program can be enhanced usingmarker-assisted selection, or marker-assisted backcrossing, of thedescendents of any cross. It is further understood that any commercialand non-commercial cultivars can be utilized in a breeding program.Factors such as, for example, emergence vigor, vegetative vigor, stresstolerance, disease resistance, branching, flowering, seed size, foragequality, and/or forage yield will generally dictate the choice.

For highly heritable traits, a choice of superior individual plantsevaluated at a single location will be effective, whereas for traitswith low heritability, selection should be based on statistical analyses(e.g., mean values) obtained from replicated evaluations of families ofrelated plants. Popular selection methods commonly include pedigreeselection, modified pedigree selection, mass selection, and recurrentselection. In some embodiments a backcross or recurrent breeding programis undertaken.

The complexity of inheritance influences choice of the breeding method.Backcross breeding can be used to transfer one or a few favorable genesfor a highly heritable trait into a desirable cultivar. This approachhas been used extensively for breeding disease-resistant cultivars.Various recurrent selection techniques are used to improvequantitatively inherited traits controlled by numerous genes. The use ofrecurrent selection in self-pollinating crops depends on the ease ofpollination, the frequency of successful hybrids from each pollination,and the number of hybrid offspring from each successful cross.

Breeding lines can be tested and compared to appropriate standards inenvironments representative of the commercial target area(s) for two ormore generations. The best lines are candidates as parents for newcommercial cultivars; those still deficient in traits may be used asparents for hybrids, or to produce new populations for furtherselection.

One method of identifying a superior plant is to observe its performancerelative to other experimental plants and to a widely grown standardcultivar. If a single observation is inconclusive, replicatedobservations can provide a better performance estimate. A breeder canselect and cross two or more parental lines, followed by repeated selfor sib pollinating and selection, producing many new geneticcombinations.

The development of new alfalfa lines requires the preparation andselection of alfalfa varieties, the crossing of these varieties andselection of superior hybrid crosses. The hybrid seed can be produced bymanual crosses between selected male-fertile parents or by using malesterility systems. Hybrids can be selected for certain single genetraits such as flower color, seed yield or herbicide resistance thatindicate that the seed is truly a hybrid. Additional data on parentallines, as well as the phenotype of the hybrid, influence the breeder'sdecision whether to continue with the specific hybrid cross.

Pedigree breeding and recurrent selection breeding methods can be usedto develop cultivars from breeding populations. Breeding programscombine desirable traits from two or more cultivars or variousbroad-based sources into breeding pools from which cultivars aredeveloped by selfing and selection of desired phenotypes into parentlines. These lines are used to produce new cultivars. New cultivars canbe evaluated to determine which have commercial potential.

Pedigree breeding is used commonly for the improvement ofself-pollinating crops. Two parents who possess favorable, complementarytraits are crossed to produce an F₁. An F₂ population is produced byselfing one or several F₁'s. Selection of the best individuals in thebest families is performed. Replicated testing of families can begin inthe F₄ generation to improve the effectiveness of selection for traitswith low heritability. At an advanced stage of inbreeding (i.e., F₆ andF₇), the best lines or mixtures of phenotypically similar lines aretested for potential release as new cultivars.

Backcross breeding and cross breeding have been used to transfer genesfor a simply inherited, highly heritable trait into a desirablehomozygous cultivar or inbred line, which is the recurrent parent. Thesource of the trait to be transferred is called the donor parent. Theresulting plant obtained from a successful backcrossing program isexpected to have the attributes of the recurrent parent (e.g., cultivar)and the desirable trait transferred from the donor parent. After theinitial cross, individuals possessing the phenotype of the donor parentare selected and repeatedly crossed (backcrossed) to the recurrentparent. After multiple backcrossing generations with selection, theresulting line is expected to have the attributes of the recurrentparent (e.g., cultivar) and the desirable trait transferred from thedonor parent.

Cross breeding or backcross breeding of an aluminum tolerant alfalfaplant may be conducted where the other parent (second alfalfa plant) isaluminum tolerant or the other parent is not aluminum tolerant.

Alfalfa plants generated of the invention may be generated using asingle-seed descent procedure. The single-seed descent procedure, in thestrict sense, refers to planting a segregating population, thenselecting one plant in this and each subsequent generation to self andcreate the next generation. When the population has been advanced fromthe F₂ to the desired level of inbreeding, the plants from which linesare derived will each trace to different F₂ individuals. The number ofplants in a population declines each generation due to failure of someseeds to germinate or some plants to produce at least one seed. As aresult, not all of the F₂ plants originally sampled in the populationwill be represented by a progeny when generation advance is completed.

Descriptions of other breeding methods that are commonly used fordifferent traits and crops can be found in reference texts (e.g., Fehr,Principles of Cultivar Development Vol. 1, pp. 2-3, 1987).

In one aspect of the present invention, the source of aluminum tolerancetrait for use in a breeding program is derived from a plant selectedfrom the group consisting of Altet1, Altet2, Altet3, Altet4 and aluminumtolerant descendants thereof. In another aspect, the source of thealuminum tolerance trait for use in a breeding program is derived from aplant selected from the group consisting of Altet4 and aluminum tolerantdescendants thereof.

Another aspect of the invention is directed to an inbred alfalfa plant,wherein said resistance is exhibited when said plant is in contact withaluminum. In one embodiment the inbred plant is an aluminum tolerantalfalfa plant. Also included in the invention is an alfalfa plant havinga genome, wherein said genome comprises one or more genetic lociconferring aluminum tolerance, wherein said one or more genetic lociassociated with one or more genetic markers linked thereto.

In one aspect, additional sources of aluminum tolerance for use in abreeding program can be identified by screening alfalfa germplasm foraluminum tolerance. In a yet another aspect, alfalfa plants can bescreened for aluminum tolerance by identifying germplasm exhibitingreduced aluminum toxicity relative to a control alfalfa plant afterinoculation or infection. In one aspect, alfalfa plants can be screenedfor aluminum tolerance using a method as described in Example 2. Inanother aspect, alfalfa plants can be screened for aluminum toleranceusing a method as described in Example 3.

In another aspect, additional sources of aluminum tolerance for use in abreeding program can be identified by screening with one or moremolecular markers linked to a genetic locus conferring aluminumtolerance.

In another aspect, aluminum tolerant alfalfa plants, varieties, lines orcultivars can be used in breeding programs to combine aluminum tolerancewith additional traits of interest. In one aspect, aluminum tolerancecan be combined with any additional trait, including other diseaseresistant traits, yield traits, and hay quality traits. Breedingprograms can also be used to combine aluminum tolerance with one or moredisease resistant traits. In another aspect, the traits that arecombined can be co-inherited in subsequent crosses.

The present invention also provides for parts of the aluminum tolerantalfalfa plants produced by a method of the present invention. Parts ofalfalfa plants, without limitation, include plant cells or parts ofplant cells, seed, endosperm, meristem, flower, anther, ovule, pollen,callus, flowers, stems, roots, stalks or leaves, scions, and rootstocks. In one embodiment of the present invention, the plant part is aseed.

The invention further provides for parts of an aluminum tolerant alfalfaplant having a genome, which comprises at least one genetic locus givingrise to aluminum tolerance in the alfalfa plant. In another embodiment,parts of alfalfa plants are derived from an alfalfa plant selected fromthe group consisting of Altet1, Altet2, Altet3 and Altet4, and aluminumtolerant descendants thereof.

One aspect of the invention includes a aluminum tolerant alfalfa plant,or the hay or seeds thereof, wherein the alfalfa plant, or the hay orseeds thereof, expresses one, or two, or three, or more independentlyselected desirable traits in addition to aluminum tolerance. In otheraspects of the invention, the plants bearing one or more desirabletraits in addition to aluminum tolerance display a greater than 10%, ora greater than 30%, or a greater than 60%, or a greater than 80%reduction in of aluminum toxicity symptoms relative to a non-resistantcontrol plant upon exposure to aluminum. Another aspect of the presentinvention is directed to a method of producing an aluminum tolerantalfalfa plant comprising: crossing an aluminum tolerant alfalfa plant,or a plant from an aluminum tolerant alfalfa line, cultivar or varietywith a second plant lacking aluminum tolerance but capable of donatingone or more of the aforementioned desirable traits.

EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventors to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 Generation of Plant Materials

The diploid Al-tolerant alfalfa Al-4 (Narasimhamoorthy et al., TheorAppl Genet 114:901-91, 2007b) was crossed with individual genotypes fromthe synthetic non-dormant tetraploid variety CUF 101 (Lehman et al.,Crop Sci 23 398-399, 1983) to produce seeds from Altet-1 throughAltet-4. Altet-4 (Al-tolerant) was manually crossed in the greenhousewith NECS-141 (semi-dormant breeding line developed in Iowa and derivedfrom a strain cross between 5454, Oneida VR, and Apica). A total of 185individuals from the NECS141Altet4 population were used for phenotypingand mapping. Of these, 110 F₁ progeny were derived from Altet-4 as thematernal parent and 75 individuals had NECS-141 as the maternal parent.Individual F₁ seeds were stored for 72 h at −20 C.°, scarified usingsand-paper, and planted in a germination mix in the greenhouse. Stemcuttings of individual genotypes were collected from the greenhouse andsterilized using 70% EtOH for 5 min followed by rinsing 3X withsterilized double distilled water for 5 min. All genotypes were clonallypropagated in modified MS medium (Murashige and Skoog, Physiol Plant15:473-797, 1962) containing the MS basal salt mixture (PhytoTechnologyLaboratories, product number M524) containing 2 mg/l of Indole-3-butyricacid (IBA) and 2% sucrose (Invitrogen, catalog number 15503-022) usingthe axillary and terminal meristems.

Example 2 Callus Bioassay Assay (CBA) for Evaluating Aluminum Tolerance

Individuals from the NECS141Altet4 population, NECS-141, Altet-1 throughAltet-4, and the CUF101-derived genotypes 95-608 and 95-653, wereevaluated for their Al-tolerance response using Blaydes medium (ALB) toinduce callus formation (Parrot and Bouton, 1990). Half of a single2-week-old callus was transferred to Blaydes media with Al (“ALB+” with400 μM of Al supplied by AlCl₃, pH 4.0) and the other half wastransferred to Blaydes medium without Al (“ALB−” at pH 4.0) aspreviously described (Parrot and Bouton, Crop Sci 30:387-389, 1990).Individual calli were weighed and transferred to fresh ALB+ and ALB−media at one week intervals for 8 weeks to determine the relative growthrate of each genotype. The experimental design was a randomized completeblock design with three replications, each of which consisted of fiveindividual calli per genotype per treatment.

Example 3 Whole-Plant Assay (WPA) for Evaluating Aluminum Tolerance

Vegetatively propagated alfalfa clones (stem cuttings) from eachindividual in the mapping population were used to evaluate the samegenotype across replications and treatments using a culture mediaadapted from Ma et al., (Nature 390:569-570, 1997) containing 200 μMCaCl₂, 1.4% Gelzan, either 0, 50 μM or 1 mM Al supplied as AlCl₃, and pH7.0 or 4.0 adjusted using 1 N HCl. Apical stem cuttings were rooted inleast macro salt (LMS) medium which consisted of 0.1 mM CaCl₂, 500 μMKNO₃ and 500 μM MgSO₄ and 1.2% Gelzan. Cuttings with visually uniformroot size and lateral root number were transferred to CaCl₂ medium −Aland +Al (1 mM AlCl₃). Plant evaluations using both the callus bioassayand the whole plant assay in media were conducted incontrolled-environment growth chambers at 25° C. and 18 h lightphotoperiod. Quantification of total root length, lateral root numbers,and branching was performed using the winRHIZO® software (RegentInstruments, Québec, Canada) commonly used to identify quantitativedifferences in root branching and length (Jahufer et al., Crop Sci.48:487-494, 2008; Zhu et al., Funct. Plant Biol. 32:749-762, 2005). Theabsolute root growth and ratio of root characteristics (biomass, length,and branching) after 2 weeks of growth in Al+ and Al− media were used asquantitative measurements to determine Al tolerance.

Example 4 Genotyping and QTL Identification

Genomic DNA from each individual from the NECS141Altet4 mappingpopulation was extracted separately using DNeasy™ Plant Mini Kit(QIAGEN, Cat. No. 69104, Valencia, USA). A total of 1024 EST-SSR primerpairs distributed throughout the alfalfa linkage groups (Sledge et al.,Theor Appl Genet 111:980-992, 2005) and those developed from alfalfatrichome ESTs (Mtri) were used to evaluate polymorphism between Altet-4,95-608, and NECS-141 and genotyping as previously described (Zhang etal., Plant Methods 4:19, 2008). A total of 538 polymorphic SSR primerspairs were used for genotyping and to identify any selfed progeny(exemplary primers are provided in Table 1). Genes implicated in the Altolerance response in other species, including those involved in organicacid synthesis (malate dehydrogenase, aluminum-activated malatetransporter (ALMT), citrate synthase, citrate dehydrogenase, isocitratedehydrogenase, oxalate oxidase, superoxide dismutase, acid phosphatases,peroxidases), signal transduction pathways, oxidative stress(phosphoenolpyruvate carboxylase, PEPC), and transporters (Ermolayev etal., Exp Bot 54:2745-2756, 2003; Maron et al., 2008; Tesfaye et al., Pl.Physiol. 127:1836-1844, 2001) were used to identify homologous genes inM. truncatula and to design molecular markers. Additional gene targetsfor marker development included Al responsive genes identified fromtranscript profiling in Medicago truncatula (Chandran et al., 2008).

Amplicons obtained using microsatellites were visualized and scoredusing GeneMapper™ 3.7 software. PCR reactions producing singleamplification products using primers designed from putative orthologs ofcandidate genes implicated in Al tolerance were used to identify lengthpolymorphism or sequenced with the BigDye® terminator v3.1 cyclesequencing kit (Applied Biosystems) and analyzed using an ABI3730genetic analyzer to confirm amplification of the target sequence andidentify candidate SNPs. Polymorphic amplicons segregating in thepopulation were scored as described by Hackett et al. (J Hered94:358-359, 2003). Simplex (1:1), duplex (5:1), and double simplex (3:1)markers were scored based on their segregation ratio in the populationto achieve maximum resolution on the parental linkage map. Recombinationfrequencies and clustering of markers into linkage groups (LGs) wasperformed using the software TetraploidMap (Hackett et al., Genetics159:1819-1832, 2001; Hackett et al., J. Hered. 98:727-729, 2007)previously used for mapping in alfalfa (Julier et al., BMC Plant Biol.3:9, 2003; Robins et al., Crop Sci. 48:1780-1786, 2008; Robins et al.,Crop Sci. 47:11-16, 2007). MapChart (Voorrips, J. Hered. 93:77-78, 2002)was used to construct the resulting linkage groups (LG). Intervalmapping for autotetraploid species was implemented for QTL analysis asdescribed by (Hackett et al., Genetics 159:1819-1832, 2001). The‘TetraploidMap’ software program (Hackett and Luo, J Hered 94:358-359,2003) was used for all analytical procedures for QTL interval mapping.Multiple regression analysis for each of the identified QTLs wasperformed to determine the allelic effect at each QTL region.

TABLE 1 Alfalfa genomic and trichome EST-SSRs used to genotype the mapping populations.Primer ID Reverse primer sequence Forward primer sequence 122161-41CCACGTTGTTGAACAGTGGAAATG (SEQ IDTGTAAAACGACGGCCAGTGCGAACTTGTTTCCGATGATGC (SEQ ID NO: 2) NO: 1) 1a07.aac.GAGCCATGTTGTTGGTGTTG (SEQ ID NO: 3)TGTAAAACGACGGCCAGTTTGGTTGGTGGGGTTATCAT(SEQ ID NO: 4) 5-1 1a09.ggt.TCTCTGGTCAGCACCAACTG (SEQ ID NO: 5)TGTAAAACGACGGCCAGTGCATGGTGAGAGACGTCGTA (SEQ ID NO: 6) 5-1 1b08.aga.TGGAGGGAAATGATTTAGCG (SEQ ID NO: 7)TGTAAAACGACGGCCAGTAACGAAAACGAAAACGAACG (SEQ ID NO: 8) 7-1 1b11.gtg.AACCTCCTCGACAACATTGG (SEQ ID NO: 9)TGTAAAACGACGGCCAGTACCTGGGATTGGGTTAGGAC (SEQ ID NO: 10) 6-1 1b12.ttc.GTCGTCGTAGAGTGGGGTGT(SEQ ID NO: 11)TGTAAAACGACGGCCAGTGAGTGGCCATGGATTCAAAC (SEQ ID NO: 12) 5-1 1c06.tta.CAAATGAGAGCACGTTGTGAA (SEQ ID NO: 13)TGTAAAACGACGGCCAGTATCATATTGGCTTGGTGCAA (SEQ ID NO: 14) 6-1 1c09.gat.TTTTCCATTCCCACCTACCA (SEQ ID NO: 15)TGTAAAACGACGGCCAGTTTTGGAAAACACTTGCCCAC (SEQ ID NO: 16) 6-1 1c11.tgg.TTGCCCTTTTGTCCAAGAAC (SEQ ID NO: 17)TGTAAAACGACGGCCAGTGACGAGAGTCCCATCAGAGC (SEQ ID NO: 18) 5-1 1c12.tgt.TTACGATCTGGCTTGGAACC (SEQ ID NO: 19)TGTAAAACGACGGCCAGTCTCGACCTGCACGACAATTA (SEQ ID NO: 20) 5-1 1d06.gaa.GAAGGTTTTGGGTGGTGATG (SEQ ID NO: 21)TGTAAAACGACGGCCAGTCCATGGCTCTTTCCTACCAA (SEQ ID NO: 22) 6-1 1e04.aaat.GACCGGGATTGATGGATATG (SEQ ID NO: 23)TGTAAAACGACGGCCAGTAACAAGAGATGGGAGGAAAAA (SEQ ID 4-1 NO: 24) 1e04.tatc.TGTTTCTGATCAGGGCATTG (SEQ ID NO: 25)TGTAAAACGACGGCCAGTTCTAGGTATTCGCTGGCGTT(SEQ ID NO: 26) 4-1 1e08.gat.ACTTCCTGACGGTCCTCCTT(SEQ ID NO: 27)TGTAAAACGACGGCCAGTGGCGCATAATCACCATTACC (SEQ ID NO: 28) 5-1 1e08.tttc.TCCTTCTGGACAAGAAACCG (SEQ ID NO: 29)TGTAAAACGACGGCCAGTTCCATCACGACATATTTCACTTTT(SEQ ID 4-1 NO: 30) 1f02.tat.TGATGCTGTCCTATGCCAAG (SEQ ID NO: 31)TGTAAAACGACGGCCAGTTGGAAAAGGCTTTGACTGTTG (SEQ ID NO: 32) 6-1 1f08.att.TGATGGATGCAATAGGGGAT(SEQ ID NO: 33)TGTAAAACGACGGCCAGTTGACATCATATGCACGGTCC (SEQ ID NO: 34) 6-1 1f08.tat.ATGAAGGTCATTGCAAGGCT(SEQ ID NO: 35)TGTAAAACGACGGCCAGTCTGCTGACTTCTGTCTGGCA (SEQ ID NO: 36) 6-1 1f10.ttg.AGTGCCGCTATGCTGCTATT(SEQ ID NO: 37)TGTAAAACGACGGCCAGTTTGATCCATGTAGCCAACCC (SEQ ID NO: 38) 6-1 1f11.aatt.TTGAAAAGACACGGGGAAGT(SEQ ID NO: 39)TGTAAAACGACGGCCAGTCCACAAAAGCAGATGGTTGA (SEQ ID NO: 40) 4-1 1f11.caa.TTGGTGAGAGCTGGTGATTG (SEQ ID NO: 41)TGTAAAACGACGGCCAGTTTACCGCTTTTGGATTCTGG (SEQ ID NO: 42) 5-1 1g03.gaa.TTTATCGGCGAAGAAGATCG (SEQ ID NO: 43)TGTAAAACGACGGCCAGTTCCCGCTTCACTTCACTTTC (SEQ ID NO: 44) 5-1 1g05.cata.CCCTAAATCAGGGGTTCAAA (SEQ ID NO: 45)TGTAAAACGACGGCCAGTCACTCATTGCTGAGGGCATA (SEQ ID NO: 46) 17-1 1g05.tct.TCAGAAATTCCCTCCCATTG (SEQ ID NO: 47)TGTAAAACGACGGCCAGTAAGAATGACGAAGAGGCGAA (SEQ ID NO: 48) 12-1 1h03.aatt.TGATTCAAGGATGGGAAAGC (SEQ ID NO: 49)TGTAAAACGACGGCCAGTTGTCTTCCGTGGTCTCACTG (SEQ ID NO: 50) 4-1 1h03.ata.GAGTTTCTGAATTCGCCGTC (SEQ ID NO: 51)TGTAAAACGACGGCCAGTTCGGCATCAATCATGTCATC (SEQ ID NO: 52) 9-1 1h09.aat.CGATAATTCACCCCCATGAC (SEQ ID NO: 53)TGTAAAACGACGGCCAGTCACAATCAAATGCATAGCCG (SEQ ID NO: 54) 11-1 2a03.aga.TCGAGAGCTCGGTATTCGAT(SEQ ID NO: 55)TGTAAAACGACGGCCAGTATCCAAGGGCGGTAGAAGAC (SEQ ID NO: 56) 5-1 2a03.gaa.TCGAGAGCTCGGTATTCGAT(SEQ ID NO: 57)TGTAAAACGACGGCCAGTGTGTGGAAGAGACCGGAGAA (SEQ ID NO: 58) 8-1 2a03.tga.AAGCACTCTGAGCCACCATT(SEQ ID NO: 59)TGTAAAACGACGGCCAGTTGAGGAAATTCTTGGGAGGA (SEQ ID NO: 60) 5-1 2a07.tatt.GCAGGGACGAAACCAGAATA (SEQ ID NO: 61)TGTAAAACGACGGCCAGTTTGCACTTCCACTAAATGACTTG (SEQ ID 4-1 NO: 62) 2a09.aac.CCCTCCAATCAAGAAACAGC (SEQ ID NO: 63)TGTAAAACGACGGCCAGTCCCAATTCCAAACCAGAAAA (SEQ ID NO: 64) 6-1 2a09.ttta.GACCATTGATCATGTCTCACG (SEQ ID NO: 65)TGTAAAACGACGGCCAGTCCAGATTGCTTACCAGGGAC (SEQ ID NO: 66) 4-1 2c06.ctc.AACAACCAAACTTGGCCTTG (SEQ ID NO: 67)TGTAAAACGACGGCCAGTTGGTCGAAGGAAGCAGAGAT(SEQ ID NO: 68) 8-1 2c06.gat.ACTTCCATTGCCGCTTCTAA (SEQ ID NO: 69)TGTAAAACGACGGCCAGTTGTGGCGAAGTAACGAAGAA (SEQ ID NO: 70) 6-1 2c06.tta.AAACCAATGATATCAAACTCCCTT(SEQ IDTGTAAAACGACGGCCAGTAAAAAGTCATGCTACAAATCATAAAAA (SEQ 9-1 NO: 71)ID NO: 72) 2c12.gga. AAATGGATTCGAACTCACGC (SEQ ID NO: 73)TGTAAAACGACGGCCAGTAAGAAGAAAAATGGCAGGAGG (SEQ ID 5-1 NO: 74) 2c12.tta.AGCCTCAAGCAGTCGTTGAC (SEQ ID NO: 75)TGTAAAACGACGGCCAGTGGAGGGGAGCAAATCTCTTT(SEQ ID NO: 76) 5-1 3c02.ata.ACTCGCTCCCTAGGGTTTGT(SEQ ID NO: 77)TGTAAAACGACGGCCAGTCCCCCAAATCCAAGAAGATT(SEQ ID NO: 78) 9-1 3d03.atc.TGTGAACATCAGGAGGTGGA (SEQ ID NO: 79)TGTAAAACGACGGCCAGTGTGAATGGTGGTCGTCTTCA (SEQ ID NO: 80) 5-1 3d03.cat.AACCATGCGGTGGTTAGGTA (SEQ ID NO: 81)TGTAAAACGACGGCCAGTCGTCATCATCATCATCACCA (SEQ ID NO: 82) 6-1 3d03.cat.TGAATGGAATCATGCAGAGG (SEQ ID NO: 83)TGTAAAACGACGGCCAGTAACGGGTGGTCTTGTGATTG (SEQ ID NO: 84) 7-1 3d03.tca.TTTTCGATCATGCCATTTGA (SEQ ID NO: 85)TGTAAAACGACGGCCAGTTTTGCACCAATGGGTAGTTC (SEQ ID NO: 86) 5-1 3e10.cag.AGCATTTGCAGTGCTAGGGT(SEQ ID NO: 87)TGTAAAACGACGGCCAGTACAGCAACAGCAACAACAGC (SEQ ID NO: 88) 6-1 3f10.gtt.GAAGCTATTTGGGCGAGCTT(SEQ ID NO: 89)TGTAAAACGACGGCCAGTCATTATGGCGTCATTTGATCC (SEQ ID NO: 90) 8-1 3g06.aga.GACACCGTTTTCGGTGATTT(SEQ ID NO: 91)TGTAAAACGACGGCCAGTTGAAACACGTTCCCACAAAG (SEQ ID NO: 92) 9-1 AA04GAACTATCACCTTTCCCTTGGA (SEQ IDTGTAAAACGACGGCCAGTATTCCGGTCGTCAGAATCAG (SEQ ID NO: 94) NO: 93) AA06AGCAGGTGGAAGAATTGGTG (SEQ ID NO: 95)TGTAAAACGACGGCCAGTCGCGTGTGTTTAGAGAGAGAGA (SEQ ID NO: 96) AC126014TTCTTCTTGGACTTGCACCA (SEQ ID NO: 97)TGTAAAACGACGGCCAGTTAAGGATGACCCAACCAAGC (SEQ ID NO: 98) AC155884TTCTTAGCTTGAAGGGCACG (SEQ ID NO: 99)TGTAAAACGACGGCCAGTCCATTCCTGGTTGTCAGTCC (SEQ ID NO: 100) AFct11TTGTGTGGAAAGAATAGGAA (SEQ ID NO: 101)TGTAAAACGACGGCCAGTGGACAGAGCAAAAGAACAAT(SEQ ID NO: 102) AFct45GCCATCTTTTCTTTTGCTTC (SEQ ID NO: 103)TGTAAAACGACGGCCAGTTAAAAAACGGAAAGAGTTGGTTAG (SEQ ID NO: 104) AI01TTGAAAATTGGGAACGGAAA (SEQ ID NO: 105)TGTAAAACGACGGCCAGTGTTGGAGTGGGAAATTGCAG (SEQ ID NO: 106) AJ02GGAAGAGGGAGAAGGAGATGA (SEQ IDTGTAAAACGACGGCCAGTTCAATGGCGAACACTTTCAC (SEQ ID NO: 108) NO: 107) AL111TGCAGCCAGGTGAATAACAA (SEQ ID NO: 109)TGTAAAACGACGGCCAGTCATCTGATGGTGGTGATTGG (SEQ ID NO: 110) AL64CCAATATGTCACTCCTTGCTGA (SEQ IDTGTAAAACGACGGCCAGTAGGTGGCAAGCCTAACTGAA (SEQ ID NO: 111) NO: 112) AL79TCCTCAACCAACCACTTCCT(SEQ ID NO: 113)TGTAAAACGACGGCCAGTCCCCATTGACGCATTCTTAC (SEQ ID NO: 114) AL81GTGGTGGAGAAGGAGCAATC (SEQ ID NO: 115)TGTAAAACGACGGCCAGTCAATCCTCCACCATCACCTT(SEQ ID NO: 116) AL83CGTTACCGTCACTGTCGTTG (SEQ ID NO: 117)TGTAAAACGACGGCCAGTCAAACCTGATTCCGACCCTA (SEQ ID NO: 118) AL84CTGCACCCCCTAAAAATCAA (SEQ ID NO: 119)TGTAAAACGACGGCCAGTCTCATTGCCCTTCTCACACA (SEQ ID NO: 120) AL92TGACTCTTGCATGCAGTTCC (SEQ ID NO: 121)TGTAAAACGACGGCCAGTTGCTCCTCCTCTGCTTCTTC (SEQ ID NO: 122) AL96GCCCCCTCACGTTTTTATTT(SEQ ID NO: 123)TGTAAAACGACGGCCAGTCAATTTTGGTTGGTTATGCTCA (SEQ ID NO: 124) AL97TCCCTCTTACACCTCTCATGC (SEQ IDTGTAAAACGACGGCCAGTTCTCCTTGGAATTGAACCTG (SEQ ID NO: 126) NO: 125) AL99CAGAAATTTCCATGCCAAAA (SEQ ID NO: 127)TGTAAAACGACGGCCAGTAGTTGTGGATTGGGTGAAGC (SEQ ID NO: 128) AW107AAACATCGGCTTCGGAAGTA (SEQ ID NO: 129)TGTAAAACGACGGCCAGTTTTTTGAGCAGTGTAATGGTGTAA (SEQ ID NO: 130) AW108CCATGGCGTCTACCCATTAT(SEQ ID NO: 131)TGTAAAACGACGGCCAGTTTTTTCACAGCACTGAAGAGG (SEQ ID NO: 132) AW11GACATTTGCAGACCACCATT(SEQ ID NO: 133)TGTAAAACGACGGCCAGTATTCGCAGTGAGCTGATCCT(SEQ ID NO: 134) AW123CATGTTTCCGGTTCTGGTTT(SEQ ID NO: 135)TGTAAAACGACGGCCAGTAGTCCCTGCAAAATCCCTTC (SEQ ID NO: 136) AW134TGGAAACAGCAAAACCACCT(SEQ ID NO: 137)TGTAAAACGACGGCCAGTTCCGAAATCTGAAACCAACC (SEQ ID NO: 138) AW150TCCACAAATGTCTAAAACCAACA (SEQ IDTGTAAAACGACGGCCAGTTTTTGTGTAGGGATGCAAAGG (SEQ ID NO: 139) NO: 140) AW16GTGGGGTTGGTGAGAGTGTT(SEQ ID NO: 141)TGTAAAACGACGGCCAGTATCGTCCCCACTGTGTCTTC (SEQ ID NO: 142) AW177CAGCAAAATCCAATCCTTCAG (SEQ IDTGTAAAACGACGGCCAGTTTCTCATCGTCACTCCAAAGAA (SEQ ID NO: 143) NO: 144) AW186TGCTTGAACTTTGAGTCTTGGA (SEQ IDTGTAAAACGACGGCCAGTTCTCTCCATCATCACCATCATC (SEQ ID NO: 145) NO: 146) AW193GACAGAACCTTTGCCGATTTT(SEQ IDTGTAAAACGACGGCCAGTGCACCAGCAGAGTAGAAGTAGC (SEQ ID NO: 147) NO: 148) AW196AACTCGCAGGTGTTTTATCGTT(SEQ IDTGTAAAACGACGGCCAGTAATCTCAACCGCAACAAACTCT(SEQ ID NO: 149) NO: 150) AW199CATGGAGAAGCAGAACTGGAG (SEQ IDTGTAAAACGACGGCCAGTCCAAACAACAACCAACTCTCTG (SEQ ID NO: 151) NO: 152) AW201CCGTCTTTACATGAATCCACAA (SEQ IDTGTAAAACGACGGCCAGTCACAGTCATCATCCTTGCTCTC (SEQ ID NO: 153) NO: 154) AW212GGTTAGGGTTTTGGGTTTGAA (SEQ IDTGTAAAACGACGGCCAGTGTCGAAATGGTTGCTTCTCTTT(SEQ ID NO: 155) NO: 156) AW213CATGTACGGGGATTGTTGTTTT(SEQ IDTGTAAAACGACGGCCAGTACCCTTGTGGGTTCTTCTTCTT(SEQ ID NO: 157) NO: 158) AW220TGCTGCTGTGCCGTAGTAGATA (SEQ IDTGTAAAACGACGGCCAGTGCCACAATTTTCTCATCATCAC (SEQ ID NO: 159) NO: 160) AW232AGCACTTTGTTCATCGTTCTGA (SEQ IDTGTAAAACGACGGCCAGTAAGAGAGTATCGTGGAGCCGTA (SEQ ID NO: 161) NO: 162) AW252CTTGAGAAAGCGAAGGTTTTGT(SEQ IDTGTAAAACGACGGCCAGTCTCGTTCATTAGCAGTTGCAGT(SEQ ID NO: 163) NO: 164) AW254CACATCTTCGTCATCATCTTCA (SEQ IDTGTAAAACGACGGCCAGTTATATGCTTGTTGAGGCCACTG (SEQ ID NO: 165) NO: 166) AW255TGCTTGAACTTTGAGTCTTGGA (SEQ IDTGTAAAACGACGGCCAGTTCTCTCCATCATCACCATCATC (SEQ ID NO: 167) NO: 168) AW258GAGTATCGGAAGAGGGTTGTTG (SEQ IDTGTAAAACGACGGCCAGTAATTGGAACCTATCGTTGTCGT(SEQ ID NO: 169) NO: 170) AW260GCATAGGAACCAGCTCTAATGG (SEQ IDTGTAAAACGACGGCCAGTACGAGGGATTGTTGTTTGAGAT(SEQ ID NO: 171) NO: 172) AW285CAACTGTGAACGCAAATCTCTC (SEQ IDTGTAAAACGACGGCCAGTAACGACGCTCTTCGACTACTTC (SEQ ID NO: 173) NO: 174) AW289GGTGCTTTCATTACATCCCATA (SEQ IDTGTAAAACGACGGCCAGTACGAGGCACACACTCTCTCTCT(SEQ ID NO: 175) NO: 176) AW306GTGTTCGTCGCATATCACCTC (SEQ IDTGTAAAACGACGGCCAGTGCATTTCCCTCTCTTTCCATAA (SEQ ID NO: 177) NO: 178) AW310CAATGCAAGAAACCCTAAAAGC (SEQ IDTGTAAAACGACGGCCAGTCCACTCAACCTCATCTCTCTACC (SEQ ID NO: 179) NO: 180)AW317 TTTTCGATTAGGTCGTGGATCT(SEQ IDTGTAAAACGACGGCCAGTACGCACATTTCCATTCTCATTC (SEQ ID NO: 181) NO: 182) AW325GCTTGTTGTTGTTGTTGATGCT(SEQ IDTGTAAAACGACGGCCAGTTCTGTAAGAGGGTCACTGCGTA (SEQ ID NO: 183) NO: 184) AW326GCATATCCATTCCAAGTTCATC (SEQ IDTGTAAAACGACGGCCAGTACTTTCTTCCTCATTGCTCTGC (SEQ ID NO: 185) NO: 186)AW329771 ATCCCATTCAAGGAAACACC (SEQ ID NO: 187)TGTAAAACGACGGCCAGTGGAATAATGCTGGTGGAAGC (SEQ ID NO: 188) AW334CGATGTTTGTTTGAGCTAGTGA (SEQ IDTGTAAAACGACGGCCAGTGAGAGAGAGAGAGAGCATTGAGC (SEQ ID NO: 189) NO: 190)AW347 GAACGGGTTTGCGATCTTT(SEQ ID NO: 191)TGTAAAACGACGGCCAGTCCATGTCTCTCAATCTTCGTCA (SEQ ID NO: 192) AW352ATCTCCTCGTGTATTCCTTCCA (SEQ IDTGTAAAACGACGGCCAGTACGTTCCTCCTTCATCTCGTAA (SEQ ID NO: 193) NO: 194) AW359TTCAAGGATCTGGTGATGATGA (SEQ IDTGTAAAACGACGGCCAGTGAGGAAGAGGAAGAGGAGGAAG (SEQ ID NO: 195) NO: 196) AW365TGTTGGTAATGTTCAAGCTCCA (SEQ IDTGTAAAACGACGGCCAGTCACCACTATCTCTTCCCTCACC (SEQ ID NO: 197) NO: 198) AW369AGAATTGAGACATGGCAGAGG (SEQ IDTGTAAAACGACGGCCAGTGCGCTCATCATCTTCATCTAAA (SEQ ID NO: 199) NO: 200) AW379TTCTCGAAATCTTCTGCTCTCG (SEQ IDTGTAAAACGACGGCCAGTGTCTCTCTCTATTCTCTTCCCTTTTC (SEQ ID NO: 201) NO: 202)AW389 GCAGCCTTCAAATCTCCATAAC (SEQ IDTGTAAAACGACGGCCAGTTCACTCTCTCACCAATCACCAC (SEQ ID NO: 203) NO: 204) AW64CATGTTTCCGGTTCTGGTTT(SEQ ID NO: 205)TGTAAAACGACGGCCAGTAGTCCCTGCAAAATCCCTTC (SEQ ID NO: 206) AW86TTGTTGCAGCAATTAAGGAAGA (SEQ IDTGTAAAACGACGGCCAGTATTGCCATTGCCTCTCTCAT(SEQ ID NO: 208) NO: 207) AW97ACAAAAACTCTCCCGGCTTT(SEQ ID NO: 209)TGTAAAACGACGGCCAGTCAAAACAATCAAACCAAAGATTG (SEQ ID NO: 210) AW98ATTCATCCTTGCTCGTTTCG (SEQ ID NO: 211)TGTAAAACGACGGCCAGTGATCAATTCGTGCAGAAGCA (SEQ ID NO: 212) BE105AAGGGCAAAACCGTAAAAGAGT(SEQ IDTGTAAAACGACGGCCAGTATCACCCCAAACCACATCTATC (SEQ ID NO: 213) NO: 214) BE112AGCGAGATAGATTTCACCGAAG (SEQ IDTGTAAAACGACGGCCAGTTTCATTTCATAGTTTTCCATTGC (SEQ ID NO: 215) NO: 216)BE118 TGCAAACTTCACCGAATAGATG (SEQ IDTGTAAAACGACGGCCAGTCTCCTTTGTAACGCAACAGCAG (SEQ ID NO: 217) NO: 218) BE120CATCATCCTTCATTTCCGATCT(SEQ IDTGTAAAACGACGGCCAGTTCTCACATTCACATTCCATTCC (SEQ ID NO: 219) NO: 220) BE123TTGATGGGTAAAGGAGAAGGTG (SEQ IDTGTAAAACGACGGCCAGTATCACAAGCCTCAACAGCCATA (SEQ ID NO: 221) NO: 222) BE41ACGCCTCTCTTTCCGATCTT(SEQ ID NO: 223)TGTAAAACGACGGCCAGTTCACTCACACTCAACACACAACA (SEQ ID NO: 224) BE67CACCAGCCTCTAAGCTCATTTT(SEQ IDTGTAAAACGACGGCCAGTCTCCATTCTCCATTTCAATACC (SEQ ID NO: 225) NO: 226) BE74GCACAAGCAGCCATATTGATAG (SEQ IDTGTAAAACGACGGCCAGTTACTGTCCCAATCTTCACAACG (SEQ ID NO: 227) NO: 228) BE76TGAAAGTTGAAGGATCTGGTGA (SEQ IDTGTAAAACGACGGCCAGTGAGGAAGAGGAAGAGGAGGAAG (SEQ ID NO: 229) NO: 230) BE84TGGGATACTGATTTTCTGCTTC (SEQ IDTGTAAAACGACGGCCAGTTCCGAACCCTACTTCCAAATTA (SEQ ID NO: 231) NO: 232) BE85CTGATTCGAGATTGGGATTGAT(SEQ IDTGTAAAACGACGGCCAGTTTTCCTCTTATTATTCTTTCATACCC (SEQ ID NO: 233) NO: 234)BE92 GATGAGGATGATGATGAATTGG (SEQ IDTGTAAAACGACGGCCAGTAGTTCAAACCCTTACCCTTCA (SEQ ID NO: 235) NO: 236) BF106GTTTTCCTGGATATTTGGATGG (SEQ IDTGTAAAACGACGGCCAGTTTCAATCTTCTCCTTTGATTGC (SEQ ID NO: 237) NO: 238) BF111TCAGTGAGAAGGTCGTTCATGT(SEQ IDTGTAAAACGACGGCCAGTTGAGAGAGAGTTCGTGGGTTG (SEQ ID NO: 239) NO: 240) BF119GTGATGAAGCATTGGTGATGAT(SEQ IDTGTAAAACGACGGCCAGTAATGGCGAACACTTTCACTCTT(SEQ ID NO: 241) NO: 242) BF120ATTTCAGAGGCAGATGGTGAAT(SEQ IDTGTAAAACGACGGCCAGTTAGCAAAATGGGTCAACAAGTG (SEQ ID NO: 243) NO: 244) BF132AATCCAGCTTTGGAAGACTCAA (SEQ IDTGTAAAACGACGGCCAGTTTCTTGTGGTGGTGATGAAAAC (SEQ ID NO: 245) NO: 246) BF142GTGTGTTCCCCAGTTCTCAGTT(SEQ IDTGTAAAACGACGGCCAGTCATACCCTTCAAATCCAACCAT(SEQ ID NO: 247) NO: 248) BF147GATTGTTCTTTGGTAAGCCTCA (SEQ IDTGTAAAACGACGGCCAGTACTGCAAGTGAAGAGGGAGAGA (SEQ ID NO: 249) NO: 250) BF149GCTTCTTTGGCTTTCTCTTCAA (SEQ IDTGTAAAACGACGGCCAGTCGTTTCCCTCTCTCACTCACTT(SEQ ID NO: 251) NO: 252) BF150ATCAGAAACAGAAGCATCAGCA (SEQ IDTGTAAAACGACGGCCAGTCTCCAAAACTCAAACTCAACCA (SEQ ID NO: 253) NO: 254) BF184CTAGACTTGCCGCTACTTTGG (SEQ IDTGTAAAACGACGGCCAGTCAACAATCACCACACACATTGA (SEQ ID NO: 255) NO: 256) BF215GGAAACATAGATGAAGCAGCAA (SEQ IDTGTAAAACGACGGCCAGTAGCAAGCAAAGAACAATCACAA (SEQ ID NO: 257) NO: 258) BF218TCGGATTTGGTTTTGAGTTTTC (SEQ IDTGTAAAACGACGGCCAGTCTCAGGAGGTGCTGTTCTTCTT(SEQ ID NO: 259) NO: 260) BF220TGAGTTTTCAGATTCAGCAGGA (SEQ IDTGTAAAACGACGGCCAGTATCATCGTCGTCGTGTTTATTG (SEQ ID NO: 261) NO: 262) BF225TTTTCATCTGTGCCCTGTAATG (SEQ IDTGTAAAACGACGGCCAGTTCACTCACACTCAACACACAACA (SEQ ID NO: 263) NO: 264)BF228 ATTAGAAGCTCCGTTACCGTCA (SEQ IDTGTAAAACGACGGCCAGTATAACCAACTCCAAACCACACC (SEQ ID NO: 265) NO: 266) BF24TTGAAAATTGGGAACGGAAA (SEQ ID NO: 267)TGTAAAACGACGGCCAGTGTTGGAGTGGGAAATTGCAG (SEQ ID NO: 268) BF257ATGCCAGGATGGTGATACATCT(SEQ IDTGTAAAACGACGGCCAGTGGATTTGGGCGTGAGACTATAC (SEQ ID NO: 269) NO: 270) BF26TCAAAGTTGTTGTTCTGCTTGAA (SEQ ID NO: 271)TGTAAAACGACGGCCAGTTCTCACACCCCAAAAACACA (SEQ ID NO: 272) BF56TCAAAGTTGTTGTTCTGCTTGAA (SEQ ID NO: 273)TGTAAAACGACGGCCAGTTCTCACACCCCAAAAACACA (SEQ ID NO: 274) BF65AAGAGCAGAAGAAGGTTTGTCG (SEQ IDTGTAAAACGACGGCCAGTACCTAAGCAAGCAAGGCAAA (SEQ ID NO: 275) NO: 276) BF71CGGTGAAATGGTGGAAGAAG (SEQ ID NO: 277)TGTAAAACGACGGCCAGTTAACAAAACCCAACCCCATC (SEQ ID NO: 278) BF79GGTGTGGAGAGGGAGGGTAG (SEQ ID NO: 279)TGTAAAACGACGGCCAGTCGAGGGATATTCTTTCCCTTAAA (SEQ ID NO: 280) BF97CTACCTCCAGCAGAACCATGTC (SEQ IDTGTAAAACGACGGCCAGTGTAACCATCCTTTGAGTTCGTCTG (SEQ ID NO: 281) NO: 282)BG115 TGCATTTGTTAACGAGTGTGAA (SEQ IDTGTAAAACGACGGCCAGTCCACAGAAGAAAGAAGAACTTGC (SEQ ID NO: 283) NO: 284)BG119 TCGAGGCCAATAGAAGACCTAA (SEQ IDTGTAAAACGACGGCCAGTGGTTCTCTTCCAATCCCTTCTT(SEQ ID NO: 285) NO: 286) BG134TTTTCAAGGAGGAGAAGATCCA (SEQ IDTGTAAAACGACGGCCAGTACCCCACCTAACCCTCTACAGT(SEQ ID NO: 287) NO: 288) BG142TGTGGTGAAGAAACGGATAGAA (SEQ IDTGTAAAACGACGGCCAGTAGTATCAATCTTTGGCGCTACC (SEQ ID NO: 289) NO: 290) BG143GGTAATCGTTGGCGTTGTTTAT(SEQ IDTGTAAAACGACGGCCAGTTCAGGTAGTTGACGACGAAGAA (SEQ ID NO: 291) NO: 292) BG157CAACGCCTCCTCTTTCTCTGTA (SEQ IDTGTAAAACGACGGCCAGTCTCAAAACCCTAACTTCTTCAACC (SEQ ID NO: 293) NO: 294)BG166 CAACTGTGAACGCAAATCTCTC (SEQ IDTGTAAAACGACGGCCAGTAACGACGCTCTTCGACTACTTC (SEQ ID NO: 295) NO: 296) BG171GGATCCAACCGAATTTCTTTC (SEQ IDTGTAAAACGACGGCCAGTACCTAGCAACCCAAATCAGAAG (SEQ ID NO: 297) NO: 298) BG172CCTCGAAAAGATTACCGAACAC (SEQ IDTGTAAAACGACGGCCAGTCGCCTTCTTCTTCAACACACTA (SEQ ID NO: 299) NO: 300) BG178TTCTCCTTGACCAACCTTGATT(SEQ IDTGTAAAACGACGGCCAGTACCCACTCAACTCAACACACAC (SEQ ID NO: 301) NO: 302) BG180AGAAGGTGGAACACGTCTCTTC (SEQ IDTGTAAAACGACGGCCAGTCTACAAGCCCAGATTTCAAAGG (SEQ ID NO: 303) NO: 304) BG181TTCGCAGTTCTTGAGTAGGTCA (SEQ IDTGTAAAACGACGGCCAGTTACTTCATGTACCCCACAACCA (SEQ ID NO: 305) NO: 306) BG186TTGTCGATGAGTTCAACGTTTC (SEQ IDTGTAAAACGACGGCCAGTACAACAAAACACAATGGGTGAC (SEQ ID NO: 307) NO: 308) BG208AGTAACCGCGAACCAAAGAGTA (SEQ IDTGTAAAACGACGGCCAGTACACCTCGAACAAGATTCATCC (SEQ ID NO: 309) NO: 310) BG218ACCATATCCACAGGCATAATCC (SEQ IDTGTAAAACGACGGCCAGTAATCCATACTCAAACCCACCAG (SEQ ID NO: 311) NO: 312) BG222ATCACGAGAACCGCCATAAGAT(SEQ IDTGTAAAACGACGGCCAGTAGGGCTGATGAGGTGGATAAT(SEQ ID NO: 313) NO: 314) BG229GAACGGGTTTGCGATCTTT(SEQ ID NO: 315)TGTAAAACGACGGCCAGTCCATGTCTCTCAATCTTCGTCA (SEQ ID NO: 316) BG231GCATGTATGATTTACAGCTCCAAG (SEQ IDTGTAAAACGACGGCCAGTCCACAGTTTCATTTTCTGTCCA (SEQ ID NO: 317) NO: 318) BG232TGCCTTTGATTAGTGCTGACAT(SEQ IDTGTAAAACGACGGCCAGTCTCTGCTCCCATCTACTTCACA (SEQ ID NO: 319) NO: 320) BG234GCAACATACCATCCCCTAAAAG (SEQ IDTGTAAAACGACGGCCAGTGCTGGAATACACCAAGCATGA (SEQ ID NO: 321) NO: 322) BG248ACATAAGCGACTGGAACAAACC (SEQ IDTGTAAAACGACGGCCAGTGGATACAAAATCCACAAGCACA (SEQ ID NO: 323) NO: 324) BG249ACATAAGCGACTGGAACAAACC (SEQ IDTGTAAAACGACGGCCAGTGGATACAAAATCCACAAGCACA (SEQ ID NO: 325) NO: 326) BG257ATTTCAGAGGCAGATGGTGAAT(SEQ IDTGTAAAACGACGGCCAGTTAGCAAAATGGGTCAACAAGTG (SEQ ID NO: 327) NO: 328) BG272CAGGGGAATCAATCAGTCAAAG (SEQ IDTGTAAAACGACGGCCAGTAAACAGAGAGACAGGAATTTGGA (SEQ ID NO: 329) NO: 330)BG280 TGTTGAAGTTGGAGTTTTGGTG (SEQ IDTGTAAAACGACGGCCAGTTCAGCAGTTAGTTTTGGTATGC (SEQ ID NO: 331) NO: 332) BG281GGTTGGAAACAAAGTCAGAACC (SEQ IDTGTAAAACGACGGCCAGTACATCATCAACAGCAAAACCAG (SEQ ID NO: 333) NO: 334) BG285TGCTTCTTGGTTTCTCATCATC (SEQ IDTGTAAAACGACGGCCAGTATGGTTATGTGGGTTGTGTTCA (SEQ ID NO: 335) NO: 336) BG82TTCCCATATGCAACAGACCTT(SEQ IDTGTAAAACGACGGCCAGTAACGGTGGTGTGTTTATTGCT(SEQ ID NO: 337) NO: 338) BG89GGCAGGAACAGATCCTTGAA (SEQ ID NO: 339)TGTAAAACGACGGCCAGTCGTAAACAAAGAAAAGCTTGAGAG (SEQ ID NO: 340) BG96TTAACGAGGGTGGTGATGGT(SEQ ID NO: 341)TGTAAAACGACGGCCAGTTCGATGTTATGGTAGCAGCAA (SEQ ID NO: 342) BI107AGCAGTGATGTCTTGGCTATGT(SEQ IDTGTAAAACGACGGCCAGTGTTTCCGGTTCTTTGTCGTTC (SEQ ID NO: 343) NO: 344) BI113AACATCGTAATGAGGAGGAGGA (SEQ IDTGTAAAACGACGGCCAGTACAGTATCAGCAACACCAGCAG (SEQ ID NO: 345) NO: 346) BI116TCAACCCTTCAGATTTTCTTCC (SEQ IDTGTAAAACGACGGCCAGTCACACTTTCTCGTTTGCTCTCT(SEQ ID NO: 347) NO: 348) BI122CAATTTCCTTAGTGGCCGTTAC (SEQ IDTGTAAAACGACGGCCAGTTTATTAGCTGGGCTTTTCTTCG (SEQ ID NO: 349) NO: 350) BI68ATCAGCGTAAATTCTGGCCTTA (SEQ IDTGTAAAACGACGGCCAGTCCATTCCAATCCACACTATCG (SEQ ID NO: 351) NO: 352) BI75CGTAGGAAGAAGGATCGAGTTC (SEQ IDTGTAAAACGACGGCCAGTCCCAATTCAAAACGAAGAACC (SEQ ID NO: 353) NO: 354) BI86CGTCGAAGTCAAAATCAATCTC (SEQ IDTGTAAAACGACGGCCAGTGAAAAGAAATCACCCCGAAGAT(SEQ ID NO: 355) NO: 356) BI96CTCATTCACCCAACCAAAATGT(SEQ IDTGTAAAACGACGGCCAGTGGCTAATTCACCTGTTTCTGCT(SEQ ID NO: 357) NO: 358) BI98TCAACAGCCAACTCAAAGTGAT(SEQ IDTGTAAAACGACGGCCAGTCATCAATCAACCCTTTCGTTTC (SEQ ID NO: 359) NO: 360)MSCWSNP0386 TTAGAGATGGTAATTGCAGTGGAC (SEQ IDTTGGTGGAAGTCATGTTTGG (SEQ ID NO: 362) NO: 361) MSCWSNP0406AACAGGACTGTGTTGCACGTA (SEQ ID CTGCTTCTGCTGATGGACAA (SEQ ID NO: 364)NO: 363) MSCWSNP0407 CCCACTGAGGGTACTCATGC (SEQ ID NO: 365)AGCTGCAACAACTCCTCCAT(SEQ ID NO: 366) MSCWSNP0453GAAACTCAAAGGGCGATCACT(SEQ ID AAGCGATATCAGAGGGTGGA (SEQ ID NO: 368)NO: 367) Mstir10581 CCTTGGCAGCTACAGGTACAG (SEQ IDTGTAAAACGACGGCCAGTGTCTGCTGCTCCAGCTAAGAA (SEQ ID NO: 369) NO: 370)Mstir10584 TCACATCAGCCCTAACATTCC (SEQ IDTGTAAAACGACGGCCAGTCCAAATATCTTCGCTCTTCCA (SEQ ID NO: 371) NO: 372)Mstir10649 GGATATCCTGGTGGAGGGTAA (SEQ IDTGTAAAACGACGGCCAGTACAACCCCATTTCCAACTTTC (SEQ ID NO: 373) NO: 374)Mstir10665 CCTCCAGGTCTAAGTCCCATT(SEQ IDTGTAAAACGACGGCCAGTCCAATGCAGTTCGGTAATCC (SEQ ID NO: 376) NO: 375)Mstir10801 GGAGCAAACATTCTACCACCA (SEQ IDTGTAAAACGACGGCCAGTTCACAAAACAAACCCTTCTTCT(SEQ ID NO: 377) NO: 378)Mstir11087 TGACTTAGACACCACCGGAGT(SEQ IDTGTAAAACGACGGCCAGTTCATCCATTCATTAAAACGCA (SEQ ID NO: 379) NO: 380)Mstir11314 ATACACCATAGCACGAGACGC (SEQ IDTGTAAAACGACGGCCAGTTAATTCGAGGAGGATTGTGGA (SEQ ID NO: 381) NO: 382)Mstir11442 GGATCCATTACCAGACAGTGC (SEQ IDTGTAAAACGACGGCCAGTTGATTTCACTTTAGCATCTTGTG (SEQ ID NO: 383) NO: 384)Mstir11470 GGAGATGAAGAAGGAGATGGG (SEQ IDTGTAAAACGACGGCCAGTTTGAAATAGTGCAAGAAGAACCC (SEQ ID NO: 385) NO: 386)Mstir11523 TGTCACTTGTTCTGGTCCTTCT(SEQ IDTGTAAAACGACGGCCAGTGGAGAGAGCAAAGTCTCTTCAA (SEQ ID NO: 387) NO: 388)Mstir11989 CAGGAACATAACTGTGACCCG (SEQ IDTGTAAAACGACGGCCAGTTCCTAATACCCCATTCATTGGT(SEQ ID NO: 389) NO: 390)Mstir12038 GCCTTTAGGCCAATCAGAGAC (SEQ IDTGTAAAACGACGGCCAGTAAGATTAGGGTTTGAGTAAGGGAA (SEQ ID NO: 391) NO: 392)Mstir7231 ACATCTTCTGGAAGACCCGTT(SEQ IDTGTAAAACGACGGCCAGTGGTAGTACTTCCTTCACTCTTCT(SEQ ID NO: 393) NO: 394)Mstir7729 ATCTGGGAAGTGTGACCTCCT(SEQ IDTGTAAAACGACGGCCAGTTCAAAACCTTGGTGTTGGTTG (SEQ ID NO: 395) NO: 396)Mstir7771 CATACTATGGTGGTGGTTGGG (SEQ IDTGTAAAACGACGGCCAGTCTCTTTAAGATTGCTTCTCTTGC (SEQ ID NO: 397) NO: 398)Mstir8491 GGACGGTTTCGAACTTCTAGC (SEQ IDTGTAAAACGACGGCCAGTCGAGGCATCTTCATCTTCAAC (SEQ ID NO: 399) NO: 400)Mstir8637 GATAAAGCTCCCACAGTTCCC (SEQ IDTGTAAAACGACGGCCAGTCTCTTTTCTCTTCAATTTTCAAT(SEQ ID NO: 401) NO: 402)Mstir8931 TACAGTTGCCCATACAGGAGG (SEQ IDTGTAAAACGACGGCCAGTCAAACAGGTGACGAGGTGAAT(SEQ ID NO: 403) NO: 404)Mstir9329 ATCAAGATCGACTGAACCACG (SEQ IDTGTAAAACGACGGCCAGTTTGGCTTTGATTGCTTCAACT(SEQ ID NO: 405) NO: 406)Mstir9849 TGAGGCTTAACCTTAGGAGGC (SEQ IDTGTAAAACGACGGCCAGTTTTCAAATCCAAGTGGTGGAG (SEQ ID NO: 407) NO: 408)Mstri10127 GGGAAACCATTTCGTACCCTA (SEQ IDTGTAAAACGACGGCCAGTAATTCCCAATTCTCATTCGTG (SEQ ID NO: 409) NO: 410)Mstri10235 TTGCCATCGTAGAAAATGGTC (SEQ IDTGTAAAACGACGGCCAGTCCTTAACACATTTTTGCTTCA (SEQ ID NO: 411) NO: 412)Mstri10456 TGTCGTCTTTTGACCATTTCC (SEQ IDTGTAAAACGACGGCCAGTTTATCATGTGCAGACAATACC (SEQ ID NO: 413) NO: 414)Mstri10592 GATTAAACATACATGCAACATTGA (SEQ IDTGTAAAACGACGGCCAGTGGTTGAAATCGACATGAGAGG (SEQ ID NO: 415) NO: 416)Mstri10686 CCAACACTTTAAGCCTCCAAA (SEQ IDTGTAAAACGACGGCCAGTTGTTCTCCTCTCTTCGTCTCTTG (SEQ ID NO: 417) NO: 418)Mstri10743 CCGGTTCTGTTTGGTAGTGAA (SEQ IDTGTAAAACGACGGCCAGTAACCAGAGAAAAATCCAACCA (SEQ ID NO: 419) NO: 420)Mstri10866 CCTTAGGCACATTGAAAACCA (SEQ IDTGTAAAACGACGGCCAGTTAAGGGTTCATGCTCACCATC (SEQ ID NO: 421) NO: 422)Mstri11061 AACATGCACAATTAAGCATTCAA (SEQ IDTGTAAAACGACGGCCAGTACCTGAAAGGCCACAAAAGAT(SEQ ID NO: 423) NO: 424)Mstri11067 AATTCGGGTGGAATAACAAGC (SEQ IDTGTAAAACGACGGCCAGTTTGCCTCGGATTATTACTTGTG (SEQ ID NO: 425) NO: 426)Mstri11090 GCAATCACCTTAGCATTTTGG (SEQ IDTGTAAAACGACGGCCAGTGCCAGTTTTGGGCAATTTTAT(SEQ ID NO: 427) NO: 428)Mstri11131 GTTCAAGCATGGAAAGTTTGG (SEQ IDTGTAAAACGACGGCCAGTGGGACCTAATATGATGAACTTACA (SEQ ID NO: 429) NO: 430)Mstri11311 TGACAGTTTCCACAATCCTCC (SEQ IDTGTAAAACGACGGCCAGTGACGAACTCTTTTCTTTTCTGACA (SEQ ID NO: 431) NO: 432)Mstri11419 ACAAGAAGAAGATTGCGACGA (SEQ IDTGTAAAACGACGGCCAGTTGAAGGAAGAAGGAAGAAGGAA (SEQ ID NO: 433) NO: 434)Mstri11460 AATTTGGACTTTGATTGTGCG (SEQ IDTGTAAAACGACGGCCAGTCAAGAACCAGATCATCAACAACA (SEQ ID NO: 435) NO: 436)Mstri11539 AAATTTCTTTCCATTGGCTCC (SEQ IDTGTAAAACGACGGCCAGTTTCATGAATTTGCTTCTATTGCAT(SEQ ID NO: 437) NO: 438)Mstri11701 AGCTTTTTCAACGAGTTCAGC (SEQ IDTGTAAAACGACGGCCAGTTTTCATCAACATCAAACACCG (SEQ ID NO: 439) NO: 440)Mstri11744 TTCTTGGCTTCGACTTCTTCA (SEQ IDTGTAAAACGACGGCCAGTCCGATTGGACTCGGAACTT(SEQ ID NO: 442) NO: 441)Mstri11748 GGATTTCGTTTGGGTTCATTT(SEQ IDTGTAAAACGACGGCCAGTTCTGTAACACAGGCAGAGTCG (SEQ ID NO: 443) NO: 444)Mstri7274 CACACATCAAAGCCCCTAAAA (SEQ IDTGTAAAACGACGGCCAGTACTCCATCAACTGGTTCACCG (SEQ ID NO: 445) NO: 446)Mstri7698 CAGTTGATGCATAGAAACGCA (SEQ IDTGTAAAACGACGGCCAGTAAGCGATTTCATTAGTAGTTGT(SEQ ID NO: 447) NO: 448)Mstri7807 TCACCAGCACATGAATCAAAA (SEQ IDTGTAAAACGACGGCCAGTAACAACCTAGATTTTCTCGACC (SEQ ID NO: 449) NO: 450)Mstri8119 AGGGTTGATGCAGATGTTACG (SEQ IDTGTAAAACGACGGCCAGTATTGCAATCATCTTCTCCCCT(SEQ ID NO: 451) NO: 452)Mstri8616 AACAATATGATCTGGCATGTCG (SEQ IDTGTAAAACGACGGCCAGTGGAAGATCACCATTTTGTCCA (SEQ ID NO: 453) NO: 454)Mstri8733 AGGTACAAGCCATGATGTCCA (SEQ IDTGTAAAACGACGGCCAGTTTTCCAAACTTTCCTTCTTTTG (SEQ ID NO: 455) NO: 456)Mstri8791 ACAAGAAGAAGATTGCGACGA (SEQ IDTGTAAAACGACGGCCAGTTGAAGGAAGAAGGAAGAAGGAA (SEQ ID NO: 457) NO: 458)Mstri8899 CGCAGCACATGTAACTTGAAA (SEQ IDTGTAAAACGACGGCCAGTCACATTCTCTTCGTGCCCTC (SEQ ID NO: 460) NO: 459)Mstri8923 TCCGAAAAAGGTGACAGATTG (SEQ IDTGTAAAACGACGGCCAGTGGCTCACAACAACAACAAAAT(SEQ ID NO: 461) NO: 462)Mstri8930 CCAAACAGATCTAAAGTTCCCA (SEQ IDTGTAAAACGACGGCCAGTTGCTTGATTATTGCTAATCGG (SEQ ID NO: 463) NO: 464)Mstri8949 TAAATGCAAGGTAGGTGGTGG (SEQ IDTGTAAAACGACGGCCAGTCGAGGACGAGTTCTGGTCAA (SEQ ID NO: 466) NO: 465)Mstri9154 AAGACCAAGAGGAATCACCGT(SEQ IDTGTAAAACGACGGCCAGTTAATTTCATTCGCGATCACAC (SEQ ID NO: 467) NO: 468)Mstri9223 TGAATGTGAGGAAGTGGGTTT(SEQ IDTGTAAAACGACGGCCAGTCCGCCTCAAATAGTTATAAACTTC (SEQ ID NO: 469) NO: 470)Mstri9326 AGTACTATTGCAATGGCGTGG (SEQ IDTGTAAAACGACGGCCAGTGGTTTCGCTTGGAATTCTGAT(SEQ ID NO: 471) NO: 472)Mstri9544 ATTTTTCCACTTCTGGTGGGA (SEQ IDTGTAAAACGACGGCCAGTCAACACAATCATTTTGGGAGC (SEQ ID NO: 473) NO: 474)Mstri9820 TCTTGTTGATATAATCTACGGAA (SEQ IDTGTAAAACGACGGCCAGTCCTGATGGTCATCACTAAGCC (SEQ ID NO: 475) NO: 476)Mstri9857 GGGACCCAATAACCGAAAATA (SEQ IDTGTAAAACGACGGCCAGTTTTGATAAACCAATCTCCCACA (SEQ ID NO: 477) NO: 478)Mt1D06 GAAGGTTTTGGGTGGTGATG (SEQ ID NO: 479)TGTAAAACGACGGCCAGTCCATGGCTCTTTCCTACCAA (SEQ ID NO: 480) Mt1G03TGGTTGATCAATGTTCCTCCT(SEQ IDTGTAAAACGACGGCCAGTAAAGAGATTGGGTCGGTGAA (SEQ ID NO: 481) NO: 482)MtBA36F01F1 AATAAACACAGATTCCAAATCCA (SEQ IDTGTAAAACGACGGCCAGTTCTTCATCGCTTTCTTCTATTTCA (SEQ ID NO: 483) NO: 484)MtBC01G06F3 TCAGGACAAACTGCCATTTC (SEQ ID NO: 485)TGTAAAACGACGGCCAGTTGCATTGAAGCAAATTAACGA (SEQ ID NO: 486) MTIC107TACGTAGCCCCTTGCTCATT(SEQ ID NO: 487)TGTAAAACGACGGCCAGTCAAACCATTTCCTCCATTGTG (SEQ ID NO: 488) MTIC124TTGGGTTGTCAATAATGCTCA (SEQ IDTGTAAAACGACGGCCAGTTTGTCACGAGTGTTGGAATTTT(SEQ ID NO: 489) NO: 490)MTIC169 GCGTGCTAGGTTTGAGAGGA (SEQ ID NO: 491)TGTAAAACGACGGCCAGTTCAAAACCCTAAAACCCTTTCTC (SEQ ID NO: 492) MTIC183TTCTCTTCAAGTGGGAGGTA (SEQ ID NO: 493)TGTAAAACGACGGCCAGTAAATGGAAGAAAGTGTCACG (SEQ ID NO: 494) MTIC19TGCAACAGAAGAAGCAAAACA (SEQ IDTGTAAAACGACGGCCAGTTCTAGAAAAAGCAATGATGTGAGA (SEQ ID NO: 495) NO: 496)MTIC233 AAGGAACAATCCCAGTTTTT(SEQ ID NO: 497)TGTAAAACGACGGCCAGTGCGTAACGTAACAACATTCA (SEQ ID NO: 498) MTIC238CCTTAGCCAAGCAAGTAAAA (SEQ ID NO: 499)TGTAAAACGACGGCCAGTTTCTTCTTCTAGGAATTTGGAG (SEQ ID NO: 500) MTIC247TGAGAGCATTGATTTTTGTG (SEQ ID NO: 501)TGTAAAACGACGGCCAGTTTCGCAGAACCTAAATTCAT(SEQ ID NO: 502) MTIC248GGATTGTGATGAAGAAATGG (SEQ ID NO: 503)TGTAAAACGACGGCCAGTTATCTCCCTTCTCCTTCTCC (SEQ ID NO: 504) MTIC249GTGGGTGAGGATGTGTGTAT(SEQ ID NO: 505)TGTAAAACGACGGCCAGTTAGGTCATGGCTATTGCTTC (SEQ ID NO: 506) MTIC250CGTTGATGATGTTCTTGATG (SEQ ID NO: 507)TGTAAAACGACGGCCAGTGCCTGAACTATTGTGAATGG (SEQ ID NO: 508) MTIC258TGAAATTCACATCAACTGGA (SEQ ID NO: 509)TGTAAAACGACGGCCAGTCACCACCTTCACCTAAGAAA (SEQ ID NO: 510) MTIC304AGCGTAAAGTAAAACCCTTTC (SEQ IDTGTAAAACGACGGCCAGTTTGGGCTTAATTTGACTGAT(SEQ ID NO: 512) NO: 511) MTIC332GGTCATACGAGCTCCTCCAT(SEQ ID NO: 513)TGTAAAACGACGGCCAGTCCCTGGGTTTTTGATCCAG (SEQ ID NO: 514) MTIC338CATTGGTGGACGAGGTCTCT(SEQ ID NO: 515)TGTAAAACGACGGCCAGTTCCCCTTAAGCTTCACTCTTTTC (SEQ ID NO: 516) MTIC343CCATTGCGGTGGCTACTCT(SEQ ID NO: 517)TGTAAAACGACGGCCAGTTCCGATCTTGCGTCCTAACT(SEQ ID NO: 518) MTIC35GGCAGGAACAGATCCTTGAA (SEQ ID NO: 519)TGTAAAACGACGGCCAGTGAAGAAGAAAAAGAGATAGATCTGTGG (SEQ ID NO: 520) MTIC354AACCTACGCTAGGGTTGCAG (SEQ ID NO: 521)TGTAAAACGACGGCCAGTAAGTGCCAAAGAACAGGGTTT(SEQ ID NO: 522) MTIC452TCACAAAAACTGCATAAAGC (SEQ ID NO: 523)TGTAAAACGACGGCCAGTCTAGTGCCAACACAAAAACA (SEQ ID NO: 524) MTIC470CCCTTCACAGAATGATTGAT(SEQ ID NO: 525)TGTAAAACGACGGCCAGTGGTTCGTGTATTTGTTCGAT(SEQ ID NO: 526) MTIC51ACAAAAACTCTCCCGGCTTT(SEQ ID NO: 527)TGTAAAACGACGGCCAGTAGTATAGTGATGAAGTGGTAGTGAACA (SEQ ID NO: 528) MTIC84GGGAAAAGGTGTAGCCATTG (SEQ ID NO: 529)TGTAAAACGACGGCCAGTTCTGAGAGAGAGACAAACAAAACAA (SEQ ID NO: 530) MTIC94CAGGGTCAGAGCAACAATCA (SEQ ID NO: 531)TGTAAAACGACGGCCAGTGCTACAACAGCGCTACATCG (SEQ ID NO: 532) MTIC95AGGAAGGAGAGGGACGAAAG (SEQ ID NO: 533)TGTAAAACGACGGCCAGTAAAGGTGTTGGGTTTTGTGG (SEQ ID NO: 534) RCS0121CTGCTTTGGTTTGGAAGAAA (SEQ ID NO: 535)TGTAAAACGACGGCCAGTGGAAAGAATATGCAATTTCTCGAT(SEQ ID NO: 536) RCS1209TGAACTTTGAAGCCACATTGA (SEQ IDTGTAAAACGACGGCCAGTAAAATCCAGAAGCACGAGTGA (SEQ ID NO: 537) NO: 538)RCS2510 GCCCTAAAAGTTGAAAGAGCA (SEQ IDTGTAAAACGACGGCCAGTCACGAGGGAACACTTCATCA (SEQ ID NO: 540) NO: 539) RCS2936CCAATGCAATTCGGTAATCC (SEQ ID NO: 541)TGTAAAACGACGGCCAGTCGTTATTTATCCCTCCGGGT(SEQ ID NO: 542) RCS4209TCACAATGGGCACCTAATCA (SEQ ID NO: 543)TGTAAAACGACGGCCAGTCAATTTTCGCTGACTGACCA (SEQ ID NO: 544) RCS4310GCCATTTGCTTCAACCTTGT(SEQ ID NO: 545)TGTAAAACGACGGCCAGTGCCATTGCTGGAATCGTAAT(SEQ ID NO: 546) RCS5452GGGCAAAACAGGAAATGAAA (SEQ ID NO: 547)TGTAAAACGACGGCCAGTATTCGATAAGGATGGCGATG (SEQ ID NO: 548) RCS5744TGTCGTCGTATCATTTCCGA (SEQ ID NO: 549)TGTAAAACGACGGCCAGTGGAGATATGCTCATTCCCCA (SEQ ID NO: 550) SNP1111TTGAAAGCACAAGGTTTCAGC (SEQ ID GTGACTTTGATGCCGGAGTT(SEQ ID NO: 552)NO: 551) TC105099 AGATAGGAATTTGGGTCGGG (SEQ ID NO: 553)TGTAAAACGACGGCCAGTACAACCATGATGTGGGAATG (SEQ ID NO: 554) TC106861GCAGGGCTGAGACTCCAGTA (SEQ ID NO: 555)TGTAAAACGACGGCCAGTAGCCCTGCTTTTTCTCCTCT(SEQ ID NO: 556) TC85780-1AAAGTGACATGATCCACAGG (SEQ ID NO: 557)TGTAAAACGACGGCCAGTGCTAAGAAAGCATGGGGTTGTTGG (SEQ ID NO: 558) TC96233GTGGCGTTTCAAATCCTTGT(SEQ ID NO: 559)TGTAAAACGACGGCCAGTTTGACTCAAACACACCCCAA (SEQ ID NO: 560)

Example 5 Evaluation of Aluminum Tolerance Using the Callus Bioassay

The parental clones Altet-4 and NECS-141, and 185 F₁ genotypes wereevaluated for their Al-tolerance response using Blaydes medium (ALB) aspreviously described (Parrot and Bouton, Crop Sci 30:387-389, 1990).Leaves and petioles from the individual genotypes were used for callusinduction. Half of a single 2-week old callus was transferred to Blaydesmedium with Al (+ALB, pH 4.0 with 400 μM of Al supplied by AlCl₃) andthe other half was transferred to Blaydes medium without Al (−ALB, pH4.0). Individual calli were weighed and transferred to fresh +ALB and−ALB medium at one week intervals for 8 weeks to determine the relativegrowth rate of each genotype. The experimental design for the callusbioassay using ALB medium included three replications with fiveindividual calli per genotype per replication. Al tolerance (+ALB/−ALB)was estimated using the total callus weight ratio (TCWR) of eachgenotype grown in medium +Al and −Al.

The relative growth rate of Altet-4 calli in media Al+vs. Al− wasconsistently higher than any other genotype evaluated, including theother Altet genotypes (FIG. 1). Therefore, Altet-4 was used as theAl-tolerant parent to develop the population NECS141Altet4 segregatingfor Al tolerance. The phenotypic evaluations of Al tolerance in theNECS141Altet4 population using the callus bioassay exhibited acontinuous and normal distribution consistent with polygenic inheritance(FIG. 2). The relative growth rates of the progeny ranged from 0.5to >1.7 suggesting transgressive segregation for Al tolerance in thispopulation.

Statistical analysis. Variation for Al tolerance from phenotypicevaluations in the callus bioassay and whole plant assay in media, andthe correlations between phenotyping systems were determined using SAS9.1 (SAS, Cary, N.C.). Statistical differences between genotypes weredetermined using PROC GLM and LSMEANS with genotype treated as a randomvariable and replication as fixed effect. PROC CORR was used to evaluatethe correlation between callus growth and root growth in both assays.The normality of the Al tolerance phenotypic data from the segregatingpopulation was evaluated with the Shapiro-Wilk test (Shapiro and Wilk,Biometrika 52:591-611, 1965) using PROC UNIVARIATE.

Results.

Phenotypic evaluations of Al tolerance in the mapping population usingthe callus bioassay exhibited a continuous and normal distribution basedon a Shapiro-Wilk (W) score of 0.92 (P<0.001) (FIG. 1A). The TCWR ofAltet-4 (0.91) was higher than the TCWR of NECS-141 (0.74). The meanTCWR of the F₁ progeny ranged from 0.50 to 1.70, with a population meanof 0.78 (sd=0.17) suggesting transgressive segregation for Al tolerancein this population.

Example 6 Evaluation of Aluminum Tolerance Using the Whole-Plant CultureMedia Assay

Clonally propagated alfalfa (stem cuttings) from each individual in themapping population were evaluated for Al-tolerance using the whole plantassay as previously described by Khu et al. (2011a, Crop Sci 52.doi:10.2135/cropsci2011.2105.0256). Briefly, the CaCl₂ medium contains200 μM 4 CaCl₂, 1.4% Gelzan (G3251, PhytoTechnology Laboratories), andeither 0 (pH 7 and pH 4) or 1 mM AlCl₃ (pH 4). The pH was adjusted to pH3 and pH 10.5 to obtain media with pH 4 and 7, respectively, afterautoclaving due to the lack of buffering capacity. Apical stem cuttingswere rooted in least macro salt (LMS) medium which consisted of 0.1 mMCaCl₂, 500 μM KNO₃ and 500 μM MgSO₄ and 1.2% Gelzan. Cuttings withvisually uniform root size and lateral root number were transferred toCaCl₂ medium −Al and +Al (1 mM AlCl₃). The experimental design includedfive replications with a single rooted cutting per replication andtreatment combination (pH 7 −Al, pH 4 −Al, and pH 4 +Al). The total rootlength of each clone was measured after 14 d of growth in medium with pH7 −Al, pH 4 −Al, and pH 4 +Al using the winRHIZO® software (RegentInstruments, Québec, Canada). Al tolerance (pH 4 +Al/pH 7 −Al) wasestimated using the average total root length ratio (TRLR) of eachgenotype grown in media at pH 7 −Al and pH 4 +Al.

The relative root length (pH7/pH4Al+) of Altet-4 and NECS-141 resultingfrom WPA evaluations in media (FIGS. 3 & 4) was 0.93 and 0.43,respectively. The phenotypic evaluations of Al tolerance in theNECS141Altet4 population using the WPA also exhibited a continuous andnormal distribution consistent with polygenic inheritance (FIG. 4). Therelative root length of the progeny ranged from 0.23 to >1.26 againindicating transgressive segregation for Al tolerance in this populationand confirming the ability of the assay to detect quantitativedifferences in Al tolerance. F₁ progeny individuals with higher Altolerance than the Al tolerant parent were observed in both the CBA andthe WPA in media assays suggesting that both parents may be contributingpositive alleles for Al tolerance.

Example 7 DNA Isolation and Genotyping

Genomic DNA from each F₁ progeny in the mapping population was extractedseparately using the DNeasy Plant Mini Kit (QIAGEN, Cat. No. 69104,Valencia, USA). A total of 755 SSR primer pairs from M. truncatulaEST-SSRs (Eujayl et al. Theor Appl Genet 108:414-422, 2004; Julier etal. BMC Plant Biol 3:9, 2003; Sledge et al. Theor Appl Genet111:980-992, 2005) and alfalfa genomic SSRs (Diwan et al. Theor ApplGenet 101:165-172, 2000), were used to screen for polymorphism betweenAltet-4 and NECS-141 as previously described by Zhang et al (PlantMethods 4:19, 2008). In addition to previously used SSR markers, 269 SSRprimers developed from alfalfa trichome unigene sequences were used toscreen for polymorphism between the two parents. Briefly, the total4,485 Medicago sativa trichome unigenes, consisting of 3,406 singletonsand 1,079 contigs or tentative consensus (TC) sequences, assembled fromtwo EST libraries (MS_TRI1 and MS_TRI2) of glandular trichomes isolatedfrom Medicago sativa stems and one EST library (MS_FAL_SSH) ofcold-treated Medicago falcata L. subsp. falcata leaves were downloadedfrom the TrichOME database (Dai et al. Pl Physiol 152:44-54, 2010).Candidate SSRs were identified from the downloaded unigenes using theSSRIT Perl scripts (Kantety et al. Plant Mol Biol 48:501-510, 2002) forperfect SSR identification and the Sputnik software(espressosoftware.com/sputnik/index.html; Verified Aug. 8 2011) forimperfect SSR identification as previously described (Zhang et al. PlantMethods 4:19, 2008). PCR primers were designed using Primer3 (Rozen andSkaletsky, Primer3 on the WWW for general users and for biologistprogrammers. In: Misener S, Krawetz S A (eds) Bioinformatics Methods andProtocols. Humana Press, pp 365-386, 1999) to amplify the identifiedcandidate SSR regions. PCR reactions were prepared in a 10 μl volume andcontained 20 ng of template DNA, 2.5 mM MgCl₂, 1×PCR buffer II (AppliedBiosystems, Foster City, Calif., USA), 0.15 mM dNTPs, 1.0 pmol each ofthe reverse primer with an additional 18 nucleotides from the M13forward sequencing universal primer (e.g. Schuelke Nature Biotechnol.18:233-234, 2000) appended to the 5′end, 0.25 pmol of the forward primer(see Table 5), and 0.5 U GoTaq® DNA polymerase (Promega, Madison, Wis.,USA). The M13 nucleotide sequences were labeled either with blue(6-FAM), green (HEX), yellow (NED) or red (PET) fluorescent tags. PCRproducts with different fluorescent labels and with different fragmentsizes were pooled for detection. A total of 1.6 μl of pooled PCRproducts were combined with 12 μl of deionized formamide and 0.5 μl ofGeneScan-500 LIZ internal size standard and analyzed on the ABI PRISM®3730 Genetic Analyzer (Applied Biosystems, Foster City, Calif., USA).GeneMapper 3.7 software was used to analyze the DNA amplicons and assignallele scores.

Example 8 Genotype and QTL Identification

A total of 305 primers from the total legume SSR primer pairs evaluatedwere polymorphic between the parental genotypes Altet-4 and NECS-141.212 SSR loci from Altet-4 and 226 loci from NECS-141 were captured inthe genetic linkage map, which consists of eight consensus LGsrepresenting the eight chromosomes in the alfalfa genome. The consensusmap length of Altet-4 was 826 cM and 745 cM for NECS-141. The consensusparental maps were constructed from the 32 co-segregation groups foreach parental genome. These co-segregation groups provide a more preciseview of linkage relationships among marker alleles and facilitateidentification of positive alleles for QTLs. Single factor analysis ofvariance (SF-ANOVA) and the non-parametric Kruskal-Wallis testidentified significant markers associated with Al tolerance from the CBAon LGs 1, 3, 4, 5, 6, and 8 in the NECS141Altet4 population (Table 2).Interval mapping was performed for all Al tolerance screening methodsutilized in this study. Based on phenotypic data from the CBA, wholeplant assay in media and whole plant assay in soil, Al tolerance QTLswere identified on six LGs (Table 2 & FIG. 5).

TABLE 2 Single factor analysis for Al tolerance from the callusbioassay. Average progeny callus P value P value Linkage growth ratio(Al+/Al−) Standard of of Kruskal- Markers group absent present errorANOVA Wallis test 608Altet4 population MTIC233-135 1 0.98 0.91 0.0310.037 0.043 MTIC247-130 1 0.89 0.97 0.031 0.02 0.015 MTIC19-154-2 2 0.960.90 0.031 0.026 0.035 MTIC51-146 3 1.24 0.92 0.068 0.002 0.000AW289-312 4 0.91 0.99 0.032 0.028 0.011 2c12gga5-1-171 5 0.86 0.96 0.0360.003 0.007 2c06ctc8-1-200 5 1.02 0.92 0.038 0.018 0.008 3d03atc5-1-2466 0.88 0.97 0.033 0.002 0.008 AW64-202 7 0.90 0.98 0.031 0.019 0.0101b11caa6-1-273 7 0.97 0.91 0.031 0.043 0.050 AW166-203 Un-linked 1.050.91 0.040 0.011 0.001 RCS5743-222 Un-linked 0.91 0.98 0.031 0.029 0.015RCS1812-142 Un-linked 0.97 0.89 0.031 0.007 0.005 NECS141Altet4population u-MTIC233-145 1 0.85 0.76 0.042 0.037 0.039 1c09gat6-1-211 30.87 0.76 0.041 0.026 0.008 1h09aat11-1-233 4 0.75 0.84 0.034 0.0310.009 MTIC249-125 4 1.09 0.77 0.123 0.019 0.010 2c06gat6-1-128 5 0.830.74 0.031 0.024 0.011 MTIC250-133 6 0.74 0.82 0.030 0.031 0.010BF26-306 7 0.82 0.73 0.030 0.01 0.006

TABLE 3 Al tolerance QTLs identified in the NECS141Altet4 populationbased on interval mapping from three phenotypic assays (callus bioassay,whole plant assay in media, and soil-based assay). Callus bio-assayWhole plant assay Soil-based assay LG Parents Position (cM) R^(2†)Position R² Position R² 1 Altet-4 72 (Rdmr1) 9.6 4 10.9 14 (Rdmr2) 7.7NECS-141 98 (Al50)  17.3 100 14.2 98 (Al1K) 26.9 3 Altet-4 74 25.2 4Altet-4 38 (Rdmr1) 29.7  4 (Rdmr2) 20.2 NECS-141 98 15.9 32 (Rdmr2) 20.65 Altet-4 62 14.1 6 Altet-4 102 (Rdmr2)  13.1 NECS-141 8 7.8 7 Altet-470 (Al50)  19.5 52 16.2 72 (Al1K) 9.9 R^(2†) = % variance explained;Al50 = Relative root length in whole plant assay in media (pH 7Al−/pH4Al+), with 50 μM Al; Al1K = Relative root length in whole plant assayin media (pH 7Al−/pH 4Al+) with 1 mM Al; Rdmr1: relative dry matter ofroots between limed and un-limed soil from soil-based experiment 1;Rdmr2: relative dry matter of roots between limed and un-limed soil insoil-based experiment 2.

Significant QTLs for Al tolerance on LGs 1, 3, 4, 5 and 7 wereidentified in which Altet-4 contributed the positive allele (FIG. 5). Inthe callus bioassay (“CBA”), the QTL for Al tolerance on LG-3 (74 cM)explained 25.2% of the variation in the Al tolerance response. Usinginterval mapping, a QTL for callus growth was also identified at 90 cMon LG 1 from Altet-4. This QTL explained 20.8% of the phenotypicvariation for total callus weight (“TCWR”). The allelic effect at eachAl tolerance QTL was estimated using the mean phenotypic value for allprogeny with a given allele at a particular locus and used to evaluatethe performance of individuals with a given allelic composition (FIG.6). For the Al tolerance QTL on LG-3, the allelic combination ‘Q₁₃’ at74 cM from homologous chromosomes H1 and H3 contributed by Altet-4, hashigher Al tolerance compared to all other possible allelic combinationsat this loci (FIG. 6A). A QTL for Al tolerance based on the CBA and thewhole plant assay (“WPA”) was identified on LG-7 and explains 19.5% ofthe variation observed in relative root length (Table 3 & FIG. 5I). Inthis case, Altet-4 contributes the positive alleles and ‘Q₁₃’ representsthe most desirable allelic combination (FIG. 6D). This QTL for Altolerance was significant at both Al concentrations used in the WPA(Al50 and Al1K) suggesting a potential mechanism of toleranceindependent of Al concentration.

Three Al tolerance QTLs were identified on chromosomes 1, 4, and 6 inthe NECS-141 parental linkage map. Although NECS-141 has lowerphenotypic values than Altet-4 (FIGS. 2 & 3; Table 4), positive allelesfor Al tolerance from NECS-141 were identified (FIG. 5B, 5E, 5H). Thesefindings indicate that while NECS-141 is phenotypically poor, it mayposses some alleles capable of increasing the trait value. A previousstudy in diploid alfalfa also identified Al tolerance QTLs from the Alsensitive parent (Sledge et al., Crop Sci 42:1121-1128, 2002). Othershave also identified QTL alleles enhancing the trait value from aphenotypically inferior parent (Tanksley and Nelson, Theor Appl Genet92:191-203, 1996; Ali et al., Theor Appl Genet 101:756-7662000; Lou etal., Euphytica 158:87-94, 2007). The QTL for Al tolerance from NECS-141located on chromosome 1 (98 cM) explain 26.9% of the variation inrelative root length (Table 3). The mean relative root growth with QTLgenotypes from the WPA at two different Al concentrations indicates that‘Q34’ is the most desirable allelic combination at this locus (FIGS. 7A& 7B).

TABLE 4 Al tolerance of alfalfa genotypes obtained from soil-based assayand whole plant assay in media. Genotypes Whole plant assay^(†)Soil-based assay^(‡) 95-608 0.56 (2)^(§) 0.54 (2) NECS-141 0.52 (3) 0.31(3) Altet-4 0.97 (1) 0.71 (1) ^(†)Ratio of total root length (pH 7Al−/pH 4 Al+ at 50 μM) ^(‡)Ratio of root dry matter (unlimed/limed)^(§)Rankings of genotypes based on performance, 1: most Al tolerant; 3:least Al tolerant.

Soil-based assays were performed twice in the greenhouse usingreplicates in time (experiment 1 and experiment 2). The two experimentsshowed significant covariance (data not shown) and thus each set ofexperimental data was analyzed separately. Soil-based phenotypic datafrom experiment 1 and 2 was used to identify two QTLs for Al toleranceon chromosome 1 and 4 from Altet-4 (Table 3, FIG. 5A & FIG. 5D). Inexperiment 2, QTLs for Al tolerance were identified on chromosomes 1, 4and 6 (Table 3). In all three cases, Altet-4 contributed the positiveallele for Al tolerance. The most desirable allelic combination at bothAl tolerance QTLs identified on chromosomes 4 is ‘Q23’ (FIG. 6B & FIG.6C).

All publications and patent applications cited herein are incorporatedby reference to the same extent as if each individual publication orpatent application was specifically and individually indicated to beincorporated by reference.

Although certain embodiments have been described in detail above, thosehaving ordinary skill in the art will clearly understand that manymodifications are possible in the embodiments without departing from theteachings thereof. All such modifications are intended to be encompassedwithin the invention as disclosed.

Example 9 Linkage Map Construction and QTL Analysis

Linkage and QTL analysis were performed using the TetraploidMap software(Hackett et al., J Hered 98:727-729, 2007) previously used for mappingin tetraploid alfalfa (Julier et al., BMC Plant Biol 3:9, 2003; Robinset al., Crop Sci 47 1-10, 2007; Robins et al., Crop Sci 48:1780-1786,2008) and tetraploid potato (Bradshaw et al., Theor Appl Genet116:193-211, 2008; Khu et al., Am J Potato Res 85:129-139, 2008). Theparental genotypes were determined based on the observed parent andoffspring marker score (Luo et al., Theor Appl Genet 100:1067-1073,2000). Markers were assigned to a given LG based on the location ofpreviously mapped SSR markers (Julier et al., BMC Plant Biol 3:9, 2003;Narasimhamoorthy et al., Theor Appl Genet 114:901-91, 2007b; Robins etal., Crop Sci 47 1-10, 2007) and simplex coupling linkages. The EMalgorithm was used to calculate the recombination frequency and LODscore to identify the most likely phase of markers on the same LG (Luoet al., Genetics 157:1369-1385, 2001). A simulated annealing algorithm(Hackett and Luo, J Hered 94:358-359, 2003) was used to identify themost accurate order of markers and distance between markers.

Multi-allelic SSR markers with either three or four alleles representingdifferent homologous chromosomes were used to identify F₁ genotypes thatinherited products of double reduction. Once the allelic combination ofeach F₁ genotype was identified, markers located in the interval betweenthis locus and the distal end of the chromosome were evaluated toconfirm double reduction in that F₁ genotype. The 27 F₁ genotypesresulting from double reduction identified in this study were notincluded in the corresponding linkage map and QTL analysis because areliable model for analyzing double reduction is not available (Bradshawet al., Theor Appl Genet 116:193-211, 2008).

Single-factor analysis of variance (SF-ANOVA) and interval mapping wereperformed using the TetraploidMap software as described by Hackett etal. (Genetics 159:1819-1832, 2001) and Bradshaw et al. (Theor Appl Genet116:193-211, 2008). The inheritance of each marker allele in the F₁progeny representing homologous chromosomes i and j from the parentalgenotypes were denoted using Q_(ij). For each marker allele combination,the mean value of all genotypes containing the allele was compared tothe mean value of the individuals without the allele. Amaximum-likelihood approach for fitting QTL models was evaluated withseparate means for each of the possible QTL genotypes (gametes Q₁Q₂,Q₁Q₃, Q₁Q₄, Q₂Q₃, Q₂Q₄, and Q₃Q₄) using a 2 cM window along thechromosome as previously described (Hackett, 2001). Significant QTLswere identified based on LOD scores greater than 3.0 and a thresholdvalue determined using 500 permutations. After the significant QTLs wereidentified, four models were evaluated using the simplex allele (absentQ_(i) versus present Q_(i)) and six models were evaluated for thedominant duplex allele on the pairs of homologous chromosomes (e.g.,Q₁Q₂+Q₁Q₃₊ Q₁Q₄₊ Q₂Q₃₊ Q₂Q₄ versus Q₃Q₄) and compared them using thelikelihood ratio test. Biallelic genotypes are reported for each markercombination with less than 5% missing data points. Interval mapping wasperformed using the permutation test with 500 iterations to declaresignificance (P<0.05).

A total of 257 primer pairs (Table 5) from the 1,024 legume SSR primerpairs evaluated were polymorphic between the parental genotypes Altet-4and NECS-141. For Altet-4, 283 SSR alleles were scored (Table 6). Ofthese, 198 were segregating in a 1:1 ratio (simplex) and 59 segregatedin a 5:1 ratio (duplex). Among these, 70 co-dominant SSR combinationswere identified by significant repulsion linkage and clusteringanalysis. For NECS-141, 231 SSR alleles segregated in a 1:1 ratio and 48segregated in a 5:1 ratio. Among these, a total of 64 co-dominant SSRcombinations were identified. SSR markers were used to construct linkagemaps for the eight LGs corresponding to the eight alfalfa chromosomes. Atotal of 185 SSR loci from Altet-4 and 205 loci from NECS-141 werecaptured in the parental genetic linkage maps, with 115 loci in commonbetween the two parental maps. The consensus maps covered 761 cM forAltet-4 and 721 cM for NECS-141, and included the 32 co-segregatinghomologous chromosomes (4 homologs for each of the eight chromosomes)for each parental genome (FIG. 9). Each homologous linkage groupcontained, on average, eleven SSR loci. The linkage maps generated inthis study include multi-allelic co-dominant SSR markers not previouslyincluded in any tetraploid alfalfa linkage maps (Brouwer and Osborn,Crop Sci 40:1387-1396, 1999; Julier et al., BMC Plant Biol 3:9, 2003;Robins et al., Crop Sci 47 1-10, 2007; Sledge et al., Theor Appl Genet111:980-992, 2005). Twenty-six double simplex markers (segregating in a3:1 ratio) associated with a simplex coupling linkage group wereidentified in both parental simplex LGs. Segregation distortion wasidentified in 27% of the markers scored in this population, which issimilar to levels of distortion in other alfalfa mapping studiesperformed using F₁ mapping populations (Julier et al., BMC Plant Biol3:9, 2003; Robins et al., Crop Sci 47 1-10, 2007).

TABLE 5List of SSR primer pairs used for linkage mapping and QTL identification in alfalfaPrimer ID Reverse primer sequence Forward primer sequence LGAmplicon size range 122161-41 CCACGTTGTTGAACAGTGGAAATGGCGAACTTGTTTCCGATGATGC 1 413-447 (SEQ ID NO: 1) (SEQ ID NO: 2)1a07aac5-1 GAGCCATGTTGTTGGTGTTG TTGGTTGGTGGGGTTATCAT 3 144-162(SEQ ID NO: 3) (SEQ ID NO: 4) la09ggt5-1 TCTCTGGTCAGCACCAACTGGCATGGTGAGAGACGTCGTA 4 250-252 (SEQ ID NO: 5) (SEQ ID NO: 6) 1b08aga7-1TGGAGGGAAATGATTTAGCG AACGAAAACGAAAACGAACG 8 175-190 (SEQ ID NO: 7)(SEQ ID NO: 8) 1b11caa6-1 AACCTCCTCGACAACATTGG AACTCAAACCCGAACAATGC 7254-281 (SEQ ID NO: 9) (SEQ ID NO: 561) 1b11gtg6-1 AACCTCCTCGACAACATTGGACCTGGGATTGGGTTAGGAC 7 313-328 (SEQ ID NO: 9) (SEQ ID NO: 10) 1b12ttc5-1GTCGTCGTAGAGTGGGGTGT GAGTGGCCATGGATTCAAAC 4 245-248 (SEQ ID NO: 11)(SEQ ID NO: 12) 1c06tta6-1 CAAATGAGAGCACGTTGTGAA ATCATATTGGCTTGGTGCAA 6214-265 (SEQ ID NO: 13) (SEQ ID NO: 14) 1c09gat6-1 TTTTCCATTCCCACCTACCATTTGGAAAACACTTGCCCAC 3 202-211 (SEQ ID NO: 15) (SEQ ID NO: 16)1c11tgg5-1 TTGCCCTTTTGTCCAAGAAC GACGAGAGTCCCATCAGAGC 5 116-169(SEQ ID NO: 17) (SEQ ID NO: 18) 1c12tgt5-1 TTACGATCTGGCTTGGAACCCTCGACCTGCACGACAATTA 5 100-235 (SEQ ID NO: 19) (SEQ ID NO: 20)1d06gaa6-1 GAAGGTTTTGGGTGGTGATG CCATGGCTCTTTCCTACCAA 2 189-192(SEQ ID NO: 21) (SEQ ID NO: 22) 1e04aaat4-1 GACCGGGATTGATGGATATGAACAAGAGATGGGAGGAAAAA 3 162-166 (SEQ ID NO: 23) (SEQ ID NO: 24)1e04tatc4-1 TGTTTCTGATCAGGGCATTG TCTAGGTATTCGCTGGCGTT 3 232-244(SEQ ID NO: 25) (SEQ ID NO: 26) 1e08gat5-1 ACTTCCTGACGGTCCTCCTTGGCGCATAATCACCATTACC 8 238-244 (SEQ ID NO: 27) (SEQ ID NO: 28)1e08tttc4-1 TCCTTCTGGACAAGAAACCG TCCATCACGACATATTTCACTTTT 8 342-343(SEQ ID NO: 29) (SEQ ID NO: 30) 1f02tat6-1 TGATGCTGTCCTATGCCAAGTGGAAAAGGCTTTGACTGTTG 5 321-335 (SEQ ID NO: 31) (SEQ ID NO: 32)1f08att6-1 TGATGGATGCAATAGGGGAT TGACATCATATGCACGGTCC 6 116-119(SEQ ID NO: 33) (SEQ ID NO: 34) 1f08tat6-1 ATGAAGGTCATTGCAAGGCTCTGCTGACTTCTGTCTGGCA 4 262-324 (SEQ ID NO: 35) (SEQ ID NO: 36)1f10ttg6-1 AGTGCCGCTATGCTGCTATT TTGATCCATGTAGCCAACCC 5 210-263(SEQ ID NO: 37) (SEQ ID NO: 38) 1f11aatt4-1 TTGAAAAGACACGGGGAAGTCCACAAAAGCAGATGGTTGA 6 192-195 (SEQ ID NO: 39) (SEQ ID NO: 40)1f11caa5-1 TTGGTGAGAGCTGGTGATTG TTACCGCTTTTGGATTCTGG 4 313-317(SEQ ID NO: 41) (SEQ ID NO: 42) 1g03gaa5-1 TTTATCGGCGAAGAAGATCGTCCCGCTTCACTTCACTTTC 8 155-220 (SEQ ID NO: 43) (SEQ ID NO: 44)1g05cata17-1 CCCTAAATCAGGGGTTCAAA CACTCATTGCTGAGGGCATA 2 139-173(SEQ ID NO: 45) (SEQ ID NO: 46) 1g05tct12-1 TCAGAAATTCCCTCCCATTGAAGAATGACGAAGAGGCGAA 4 268-277 (SEQ ID NO: 47) (SEQ ID NO: 48)1h03aatt4-1 TGATTCAAGGATGGGAAAGC TGTCTTCCGTGGTCTCACTG 1 202-229(SEQ ID NO: 49) (SEQ ID NO: 50) 1h03ata9-1 GAGTTTCTGAATTCGCCGTCTCGGCATCAATCATGTCATC 1 300-303 (SEQ ID NO: 51) (SEQ ID NO: 52)1h09aat11-1 CGATAATTCACCCCCATGAC CACAATCAAATGCATAGCCG 4 218-237(SEQ ID NO: 53) (SEQ ID NO: 54) 2a03aga5-1 TCGAGAGCTCGGTATTCGATATCCAAGGGCGGTAGAAGAC 4 279-284 (SEQ ID NO: 55) (SEQ ID NO: 56)2a03gaa8-1 TCGAGAGCTCGGTATTCGAT GTGTGGAAGAGACCGGAGAA 4 230-236(SEQ ID NO: 57) (SEQ ID NO: 58) 2a03tga5-1 AAGCACTCTGAGCCACCATTTGAGGAAATTCTTGGGAGGA 8 277-292 (SEQ ID NO: 59) (SEQ ID NO: 60)2a07tatt4-1 GCAGGGACGAAACCAGAATA TTGCACTTCCACTAAATGACTTG 5 316-318(SEQ ID NO: 61) (SEQ ID NO: 62) 2a09aac6-1 CCCTCCAATCAAGAAACAGCCCCAATTCCAAACCAGAAAA 8 256-282 (SEQ ID NO: 63) (SEQ ID NO: 64)2a09ttta4-1 GACCATTGATCATGTCTCACG CCAGATTGCTTACCAGGGAC 3 276-303(SEQ ID NO: 65) (SEQ ID NO: 66) 2c06ctc8-1 AACAACCAAACTTGGCCTTGTGGTCGAAGGAAGCAGAGAT 5 173-200 (SEQ ID NO: 67) (SEQ ID NO: 68)2c06gat6-1 ACTTCCATTGCCGCTTCTAA TGTGGCGAAGTAACGAAGAA 5 128-137(SEQ ID NO: 69) (SEQ ID NO: 70) 2c06tta9-1 AAACCAATGATATCAAACTCCCTTAAAAAGTCATGCTACAAATCATAAAAA 3 244-304 (SEQ ID NO: 71) (SEQ ID NO: 72)2c12gga5-1 AAATGGATTCGAACTCACGC AAGAAGAAAAATGGCAGGAGG 5 165-174(SEQ ID NO: 73) (SEQ ID NO: 74) 2c12tta5-1 AGCCTCAAGCAGTCGTTGACGGAGGGGAGCAAATCTCTTT 5 316-319 (SEQ ID NO: 75) (SEQ ID NO: 76)3d03atc5-1 TGTGAACATCAGGAGGTGGA GTGAATGGTGGTCGTCTTCA 6 206-268(SEQ ID NO: 79) (SEQ ID NO: 80) 3d03cat6-1 AACCATGCGGTGGTTAGGTACGTCATCATCATCATCACCA 6 175-181 (SEQ ID NO: 81) (SEQ ID NO: 82)3d03cat7-1 TGAATGGAATCATGCAGAGG AACGGGTGGTCTTGTGATTG 6 284-313(SEQ ID NO: 83) (SEQ ID NO: 84) 3d03tca5-1 TTTTCGATCATGCCATTTGATTTGCACCAATGGGTAGTTC 6 207-226 (SEQ ID NO: 85) (SEQ ID NO: 86)3e10cag6-1 AGCATTTGCAGTGCTAGGGT ACAGCAACAGCAACAACAGC 1 187-196(SEQ ID NO: 87) (SEQ ID NO: 88) 3f10gtt8-1 GAAGCTATTTGGGCGAGCTTCATTATGGCGTCATTTGATCC 4 190-197 (SEQ ID NO: 89) (SEQ ID NO: 90)3g06aga9-1 GACACCGTTTTCGGTGATTT TGAAACACGTTCCCACAAAG 2 295-301(SEQ ID NO: 91) (SEQ ID NO: 92) AA04 GAACTATCACCTTTCCCTTGGAATTCCGGTCGTCAGAATCAG 4 306-315 (SEQ ID NO: 93) (SEQ ID NO: 94) AA06AGCAGGTGGAAGAATTGGTG CGCGTGTGTTTAGAGAGAGAGA 5 177-179 (SEQ ID NO: 95)(SEQ ID NO: 96) AC126014 TTCTTCTTGGACTTGCACCA TAAGGATGACCCAACCAAGC 4301-308 (SEQ ID NO: 97) (SEQ ID NO: 98) AC155884 TTCTTAGCTTGAAGGGCACGCCATTCCTGGTTGTCAGTCC 2 154-162 (SEQ ID NO: 99) (SEQ ID NO: 100) AFct11TTGTGTGGAAAGAATAGGAA GGACAGAGCAAAAGAACAAT 6 203-210 (SEQ ID NO: 101)(SEQ ID NO: 102) AFct45 GCCATCTTTTCTTTTGCTTC TAAAAAACGGAAAGAGTTGGTTAG 7153-162 (SEQ ID NO: 103) (SEQ ID NO: 104) AI01 TTGAAAATTGGGAACGGAAAGTTGGAGTGGGAAATTGCAG 7 196-200 (SEQ ID NO: 105) (SEQ ID NO: 106) AJ02GGAAGAGGGAGAAGGAGATGA TCAATGGCGAACACTTTCAC 1 222-231 (SEQ ID NO: 107)(SEQ ID NO: 108) AL111 TGCAGCCAGGTGAATAACAA CATCTGATGGTGGTGATTGG 8197-200 (SEQ ID NO: 109) (SEQ ID NO: 110) AL64 CCAATATGTCACTCCTTGCTGAAGGTGGCAAGCCTAACTGAA 8 237-240 (SEQ ID NO: 111) (SEQ ID NO: 112) AL79TCCTCAACCAACCACTTCCT CCCCATTGACGCATTCTTAC 8 259-268 (SEQ ID NO: 113)(SEQ ID NO: 114) AL81 GTGGTGGAGAAGGAGCAATC CAATCCTCCACCATCACCTT 1228-257 (SEQ ID NO: 115) (SEQ ID NO: 116) AL83 CGTTACCGTCACTGTCGTTGCAAACCTGATTCCGACCCTA 1 153-159 (SEQ ID NO: 117) (SEQ ID NO: 118) AL84CTGCACCCCCTAAAAATCAA CTCATTGCCCTTCTCACACA 4 156-164 (SEQ ID NO: 119)(SEQ ID NO: 120) AL92 TGACTCTTGCATGCAGTTCC TGCTCCTCCTCTGCTTCTTC 8201-209 (SEQ ID NO: 121) (SEQ ID NO: 122) AL96 GCCCCCTCACGTTTTTATTTCAATTTTGGTTGGTTATGCTCA 8 150-155 (SEQ ID NO: 123) (SEQ ID NO: 124) AL97TCCCTCTTACACCTCTCATGC TCTCCTTGGAATTGAACCTG 6 144-194 (SEQ ID NO: 125)(SEQ ID NO: 126) AL99 CAGAAATTTCCATGCCAAAA AGTTGTGGATTGGGTGAAGC 2167-176 (SEQ ID NO: 127) (SEQ ID NO: 128) AW107 AAACATCGGCTTCGGAAGTATTTTTGAGCAGTGTAATGGTGTAA 3 203-205 (SEQ ID NO: 129) (SEQ ID NO: 130)AW108 CCATGGCGTCTACCCATTAT TTTTTCACAGCACTGAAGAGG 3 220-223(SEQ ID NO: 131) (SEQ ID NO: 132) AW11 GACATTTGCAGACCACCATTATTCGCAGTGAGCTGATCCT 8 214-237 (SEQ ID NO: 133) (SEQ ID NO: 134) AW123CATGTTTCCGGTTCTGGTTT AGTCCCTGCAAAATCCCTTC 7 200-207 (SEQ ID NO: 135)(SEQ ID NO: 136) AW134 TGGAAACAGCAAAACCACCT TCCGAAATCTGAAACCAACC 4201-227 (SEQ ID NO: 137) (SEQ ID NO: 138) AW150 TCCACAAATGTCTAAAACCAACATTTTGTGTAGGGATGCAAAGG 7 186-193 (SEQ ID NO: 139) (SEQ ID NO: 140) AW16GTGGGGTTGGTGAGAGTGTT ATCGTCCCCACTGTGTCTTC 2 207-234 (SEQ ID NO: 141)(SEQ ID NO: 142) AW177 CAGCAAAATCCAATCCTTCAG TTCTCATCGTCACTCCAAAGAA 7288-291 (SEQ ID NO: 143) (SEQ ID NO: 144) AW186 TGCTTGAACTTTGAGTCTTGGATCTCTCCATCATCACCATCATC 8 237-240 (SEQ ID NO: 145) (SEQ ID NO: 146) AW196AACTCGCAGGTGTTTTATCGTT AATCTCAACCGCAACAAACTCT 5 209-217 (SEQ ID NO: 149)(SEQ ID NO: 150) AW199 CATGGAGAAGCAGAACTGGAG CCAAACAACAACCAACTCTCTG 1318-333 (SEQ ID NO: 151) (SEQ ID NO: 152) AW201 CCGTCTTTACATGAATCCACAACACAGTCATCATCCTTGCTCTC 8 286-299 (SEQ ID NO: 153)(Nucleotides 19 through 40  of SEQ ID NO: 154) AW212GGTTAGGGTTTTGGGTTTGAA GTCGAAATGGTTGCTTCTCTTT 7 242-272 (SEQ ID NO: 155)(Nucleotides 19 through 40  of SEQ ID NO: 156) AW213CATGTACGGGGATTGTTGTTTT ACCCTTGTGGGTTCTTCTTCTT 3 262-270 (SEQ ID NO: 157)(Nucleotides 19 through 40  of SEQ ID NO: 158) AW232AGCACTTTGTTCATCGTTCTGA AAGAGAGTATCGTGGAGCCGTA 4 189-198 (SEQ ID NO: 161)(Nucleotides 19 through 40  of SEQ ID NO: 162) AW252CTTGAGAAAGCGAAGGTTTTGT CTCGTTCATTAGCAGTTGCAGT 7 142-144 (SEQ ID NO: 163)(Nucleotides 19 through 40  of SEQ ID NO: 164) AW254CACATCTTCGTCATCATCTTCA TATATGCTTGTTGAGGCCACTG 7 210-216 (SEQ ID NO: 165)(Nucleotides 19 through 40  of SEQ ID NO: 166) AW255TGCTTGAACTTTGAGTCTTGGA TCTCTCCATCATCACCATCATC 8 234-243 (SEQ ID NO: 167)(Nucleotides 19 through 40  of SEQ ID NO: 168) AW258GAGTATCGGAAGAGGGTTGTTG AATTGGAACCTATCGTTGTCGT 8 240-243 (SEQ ID NO: 169)(Nucleotides 19 through 40  of SEQ ID NO: 170) AW285CAACTGTGAACGCAAATCTCTC AACGACGCTCTTCGACTACTTC 4 119-140 (SEQ ID NO: 173)(Nucleotides 19 through 40  of SEQ ID NO: 174) AW289GGTGCTTTCATTACATCCCATA ACGAGGCACACACTCTCTCTCT 4 301-307 (SEQ ID NO: 175)(Nucleotides 19 through 40  of SEQ ID NO: 176) AW306GTGTTCGTCGCATATCACCTC GCATTTCCCTCTCTTTCCATAA 3 242-247 (SEQ ID NO: 177)(Nucleotides 19 through 40  of SEQ ID NO: 178) AW310CAATGCAAGAAACCCTAAAAGC CCACTCAACCTCATCTCTCTACC 2 327-353(SEQ ID NO: 179) (Nucleotides 19 through 41  of SEQ ID NO: 180) AW325GCTTGTTGTTGTTGTTGATGCT TCTGTAAGAGGGTCACTGCGTA 8 160-172 (SEQ ID NO: 183)(Nucleotides 19 through 40  of SEQ ID NO: 184) AW326GCATATCCATTCCAAGTTCATC ACTTTCTTCCTCATTGCTCTGC 7 199-206 (SEQ ID NO: 185)(Nucleotides 19 through 40  of SEQ ID NO: 186) AW329771ATCCCATTCAAGGAAACACC GGAATAATGCTGGTGGAAGC 7 244-254 (SEQ ID NO: 187)(Nucleotides 19 through 38  of SEQ ID NO: 188) AW334CGATGTTTGTTTGAGCTAGTGA GAGAGAGAGAGAGAGCATTGAGC 8 240-247(SEQ ID NO: 189) (Nucleotides 19 through 41  of SEQ ID NO: 190) AW347GAACGGGTTTGCGATCTTT CCATGTCTCTCAATCTTCGTCA 4 321-324 (SEQ ID NO: 191)(Nucleotides 19 through 40  of SEQ ID NO: 192) AW352ATCTCCTCGTGTATTCCTTCCA ACGTTCCTCCTTCATCTCGTAA 7 207-212 (SEQ ID NO: 193)(Nucleotides 19 through 40  of SEQ ID NO: 194) AW359TTCAAGGATCTGGTGATGATGA GAGGAAGAGGAAGAGGAGGAAG 5 175-184 (SEQ ID NO: 195)(Nucleotides 19 through 40  of SEQ ID NO: 196) AW365TGTTGGTAATGTTCAAGCTCCA CACCACTATCTCTTCCCTCACC 1 261-273 (SEQ ID NO: 197)(Nucleotides 19 through 40  of SEQ ID NO: 198) AW369AGAATTGAGACATGGCAGAGG GCGCTCATCATCTTCATCTAAA 5 103-169 (SEQ ID NO: 199)(Nucleotides 19 through 40  of SEQ ID NO: 200) AW379TTCTCGAAATCTTCTGCTCTCG GTCTCTCTCTATTCTCTTCCCTTTTC 3 165-174(SEQ ID NO: 201) (Nucleotides 19 through 44  of SEQ ID NO: 202) AW389GCAGCCTTCAAATCTCCATAAC TCACTCTCTCACCAATCACCAC 5 482-497 (SEQ ID NO: 203)(Nucleotides 19 through 40  of SEQ ID NO: 204) AW64 CATGTTTCCGGTTCTGGTTTAGTCCCTGCAAAATCCCTTC 7 200-207 (SEQ ID NO: 205)(Nucleotides 19 through 38  of SEQ ID NO: 206) AW86TTGTTGCAGCAATTAAGGAAGA ATTGCCATTGCCTCTCTCAT 1 174-222 (SEQ ID NO: 207)(Nucleotides 19 through 38  of SEQ ID NO: 208) AW97 ACAAAAACTCTCCCGGCTTTCAAAACAATCAAACCAAAGATTG 3 220-232 (SEQ ID NO: 209)(Nucleotides 19 through 41  of SEQ ID NO: 210) AW98 ATTCATCCTTGCTCGTTTCGGATCAATTCGTGCAGAAGCA 2 205-232 (SEQ ID NO: 211)(Nucleotides 19 through 38  of SEQ ID NO: 212) BE105AAGGGCAAAACCGTAAAAGAGT ATCACCCCAAACCACATCTATC 1 236-242 (SEQ ID NO: 213)(Nucleotides 19 through 40  of SEQ ID NO: 214) BE114ATGAAGCTGTTGTTGTTGCAGT CCACCTCATCACTCCGTAAAA 3 198-220 (SEQ ID NO: 562)(SEQ ID NO: 563)  BE118 TGCAAACTTCACCGAATAGATG CTCCTTTGTAACGCAACAGCAG 8233-241 (SEQ ID NO: 217) (Nucleotides 19 through 40  of SEQ ID NO: 218)BE120 CATCATCCTTCATTTCCGATCT TCTCACATTCACATTCCATTCC 5 234-234(SEQ ID NO: 219) (Nucleotides 19 through 40  of SEQ ID NO: 220) BE123TTGATGGGTAAAGGAGAAGGTG ATCACAAGCCTCAACAGCCATA 7 211-229 (SEQ ID NO: 221)(Nucleotides 19 through 40  of SEQ ID NO: 222) BE41 ACGCCTCTCTTTCCGATCTTTCACTCACACTCAACACACAACA 3 212-223 (SEQ ID NO: 223)(Nucleotides 19 through 41  of SEQ ID NO: 224) BE67CACCAGCCTCTAAGCTCATTTT CTCCATTCTCCATTTCAATACC 3 167-182 (SEQ ID NO: 225)(Nucleotides 19 through 40  of SEQ ID NO: 226) BE74GCACAAGCAGCCATATTGATAG TACTGTCCCAATCTTCACAACG 7 238-267 (SEQ ID NO: 227)(Nucleotides 19 through 40  of SEQ ID NO: 228) BE76TGAAAGTTGAAGGATCTGGTGA GAGGAAGAGGAAGAGGAGGAAG 5 182-191 (SEQ ID NO: 229)(Nucleotides 19 through 40  of SEQ ID NO: 230) BE84TGGGATACTGATTTTCTGCTTC TCCGAACCCTACTTCCAAATTA 4 223-229 (SEQ ID NO: 231)(Nucleotides 19 through 40  of SEQ ID NO: 232) BE85CTGATTCGAGATTGGGATTGAT TTTCCTCTTATTATTCTTTCATACCC 3 233-246(SEQ ID NO: 233) (Nucleotides 19 through 44  of SEQ ID NO: 234) BE92GATGAGGATGATGATGAATTGG AGTTCAAACCCTTACCCTTCA 6 190-199 (SEQ ID NO: 235)(Nucleotides 19 through 39  of SEQ ID NO: 236) BF106GTTTTCCTGGATATTTGGATGG TTCAATCTTCTCCTTTGATTGC 5 214-218 (SEQ ID NO: 237)(Nucleotides 19 through 40  of SEQ ID NO: 238) BF111TCAGTGAGAAGGTCGTTCATGT TGAGAGAGAGTTCGTGGGTTG 2 170-205 (SEQ ID NO: 239)(Nucleotides 19 through 39  of SEQ ID NO: 240) BF119GTGATGAAGCATTGGTGATGAT AATGGCGAACACTTTCACTCTT 1 119-159 (SEQ ID NO: 241)(Nucleotides 19 through 40  of SEQ ID NO: 242) BF120ATTTCAGAGGCAGATGGTGAAT TAGCAAAATGGGTCAACAAGTG 3 224-226 (SEQ ID NO: 243)(Nucleotides 19 through 40  of SEQ ID NO: 244) BF132AATCCAGCTTTGGAAGACTCAA TTCTTGTGGTGGTGATGAAAAC 7 205-214 (SEQ ID NO: 245)(Nucleotides 19 through 40  of SEQ ID NO: 246) BF142GTGTGTTCCCCAGTTCTCAGTT CATACCCTTCAAATCCAACCAT 7 263-266 (SEQ ID NO: 247)(Nucleotides 19 through 40  of SEQ ID NO: 248) BF147GATTGTTCTTTGGTAAGCCTCA ACTGCAAGTGAAGAGGGAGAGA 5 147-150 (SEQ ID NO: 249)(Nucleotides 19 through 40  of SEQ ID NO: 250) BF149GCTTCTTTGGCTTTCTCTTCAA CGTTTCCCTCTCTCACTCACTT 6 103-113 (SEQ ID NO: 251)(Nucleotides 19 through 40  of SEQ ID NO: 252) BF150ATCAGAAACAGAAGCATCAGCA CTCCAAAACTCAAACTCAACCA 2 274-277 (SEQ ID NO: 253)(Nucleotides 19 through 40  of SEQ ID NO: 254) BF184CTAGACTTGCCGCTACTTTGG CAACAATCACCACACACATTGA 4 284-304 (SEQ ID NO: 255)(Nucleotides 19 through 40  of SEQ ID NO: 256) BF215GGAAACATAGATGAAGCAGCAA AGCAAGCAAAGAACAATCACAA 2 230-237 (SEQ ID NO: 257)(Nucleotides 19 through 40  of SEQ ID NO: 258) BF218TCGGATTTGGTTTTGAGTTTTC CTCAGGAGGTGCTGTTCTTCTT 8 243-245 (SEQ ID NO: 259)(Nucleotides 19 through 40  of SEQ ID NO: 260) BF220TGAGTTTTCAGATTCAGCAGGA ATCATCGTCGTCGTGTTTATTG 3 287-308 (SEQ ID NO: 261)(Nucleotides 19 through 40  of SEQ ID NO: 262) BF223AATAGGGTTTGATTGAGGAGCA CGACGAACAGAAGCTAAGAGATG 4 124-136(SEQ ID NO: 564) (SEQ ID NO: 565)  BF225 TTTTCATCTGTGCCCTGTAATGTCACTCACACTCAACACACAACA 3 190-201 (SEQ ID NO: 263)(Nucleotides 19 through 41  of SEQ ID NO: 264) BF228ATTAGAAGCTCCGTTACCGTCA ATAACCAACTCCAAACCACACC 1 143-153 (SEQ ID NO: 265)(Nucleotides 19 through 40  of SEQ ID NO: 266) BF24 TTGAAAATTGGGAACGGAAAGTTGGAGTGGGAAATTGCAG 7 196-200 (SEQ ID NO: 267)(Nucleotides 19 through 38  of SEQ ID NO: 268) BF257ATGCCAGGATGGTGATACATCT GGATTTGGGCGTGAGACTATAC 3 412-430 (SEQ ID NO: 269)(Nucleotides 19 through 40  of SEQ ID NO: 270) BF26TCAAAGTTGTTGTTCTGCTTGAA TCTCACACCCCAAAAACACA 7 289-306 (SEQ ID NO: 271)(Nucleotides 19 through 38  of SEQ ID NO: 272) BF71 CGGTGAAATGGTGGAAGAAGTAACAAAACCCAACCCCATC 4 216-229 (SEQ ID NO: 277)(Nucleotides 19 through 38  of SEQ ID NO: 278) BF79 GGTGTGGAGAGGGAGGGTAGCGAGGGATATTCTTTCCCTTAAA 3 182-197 (SEQ ID NO: 279)(Nucleotides 19 through 41  of SEQ ID NO: 280) BF97CTACCTCCAGCAGAACCATGTC GTAACCATCCTTTGAGTTCGTCTG 8 249-252(SEQ ID NO: 281) (Nucleotides 19 through 42  of SEQ ID NO: 282) BG115TGCATTTGTTAACGAGTGTGAA CCACAGAAGAAAGAAGAACTTGC 3 208-230(SEQ ID NO: 283) (Nucleotides 19 through 41  of SEQ ID NO: 284) BG119TCGAGGCCAATAGAAGACCTAA GGTTCTCTTCCAATCCCTTCTT 7 265-281 (SEQ ID NO: 285)(Nucleotides 19 through 40  of SEQ ID NO: 286) BG134TTTTCAAGGAGGAGAAGATCCA ACCCCACCTAACCCTCTACAGT 5 190-203 (SEQ ID NO: 287)(Nucleotides 19 through 40  of SEQ ID NO: 288) BG137CAGAGCAATAAGAACACCAGGA ACTCTTCCTCGCCACTTCAAC 1 320-323 (SEQ ID NO: 566)(SEQ ID NO: 567) BG143 GGTAATCGTTGGCGTTGTTTAT TCAGGTAGTTGACGACGAAGAA 2125-134 (SEQ ID NO: 291) (Nucleotides 19 through 40  of SEQ ID NO: 292)BG157 CAACGCCTCCTCTTTCTCTGTA CTCAAAACCCTAACTTCTTCAACC 5 146-154(SEQ ID NO: 293) (Nucleotides 19 through 42  of SEQ ID NO: 294) BG166CAACTGTGAACGCAAATCTCTC AACGACGCTCTTCGACTACTTC 4 120-141 (SEQ ID NO: 295)(Nucleotides 19 through 40  of SEQ ID NO: 296) BG171GGATCCAACCGAATTTCTTTC ACCTAGCAACCCAAATCAGAAG 4 192-195 (SEQ ID NO: 297)(Nucleotides 19 through 40  of SEQ ID NO: 298) BG172CCTCGAAAAGATTACCGAACAC CGCCTTCTTCTTCAACACACTA 4 190-194 (SEQ ID NO: 299)(Nucleotides 19 through 40  of SEQ ID NO: 300) BG178TTCTCCTTGACCAACCTTGATT ACCCACTCAACTCAACACACAC 7 212-226 (SEQ ID NO: 301)(Nucleotides 19 through 40  of SEQ ID NO: 302) BG180AGAAGGTGGAACACGTCTCTTC CTACAAGCCCAGATTTCAAAGG 1 159-172 (SEQ ID NO: 303)(Nucleotides 19 through 40  of SEQ ID NO: 304) BG181TTCGCAGTTCTTGAGTAGGTCA TACTTCATGTACCCCACAACCA 1 162-167 (SEQ ID NO: 305)(Nucleotides 19 through 40  of SEQ ID NO: 306) BG186TTGTCGATGAGTTCAACGTTTC ACAACAAAACACAATGGGTGAC 8 166-189 (SEQ ID NO: 307)(Nucleotides 19 through 40  of SEQ ID NO: 308) BG208AGTAACCGCGAACCAAAGAGTA ACACCTCGAACAAGATTCATCC 1 220-226 (SEQ ID NO: 309)(Nucleotides 19 through 40  of SEQ ID NO: 310) BG218ACCATATCCACAGGCATAATCC AATCCATACTCAAACCCACCAG 2 285-301 (SEQ ID NO: 311)(Nucleotides 19 through 40  of SEQ ID NO: 312) BG222ATCACGAGAACCGCCATAAGAT AGGGCTGATGAGGTGGATAAT 4 228-237 (SEQ ID NO: 313)(Nucleotides 19 through 39  of SEQ ID NO: 314) BG229 GAACGGGTTTGCGATCTTTCCATGTCTCTCAATCTTCGTCA 4 321-323 (SEQ ID NO: 315)(Nucleotides 19 through 40  of SEQ ID NO: 316) BG231GCATGTATGATTTACAGCTCCAAG CCACAGTTTCATTTTCTGTCCA 2 383-399(SEQ ID NO: 317) (Nucleotides 19 through 40  of SEQ ID NO: 318) BG232TGCCTTTGATTAGTGCTGACAT CTCTGCTCCCATCTACTTCACA 8 167-172 (SEQ ID NO: 319)(Nucleotides 19 through 40  of SEQ ID NO: 320) BG234GCAACATACCATCCCCTAAAAG GCTGGAATACACCAAGCATGA 1 217-251 (SEQ ID NO: 321)(Nucleotides 19 through 40  of SEQ ID NO: 322) BG248ACATAAGCGACTGGAACAAACC GGATACAAAATCCACAAGCACA 1 284-348 (SEQ ID NO: 323)(Nucleotides 19 through 40  of SEQ ID NO: 324) BG257ATTTCAGAGGCAGATGGTGAAT TAGCAAAATGGGTCAACAAGTG 3 223-230 (SEQ ID NO: 327)(Nucleotides 19 through 40  of SEQ ID NO: 328) BG272CAGGGGAATCAATCAGTCAAAG AAACAGAGAGACAGGAATTTGGA 3 446-456(SEQ ID NO: 329) (Nucleotides 19 through 41  of SEQ ID NO: 330) BG280TGTTGAAGTTGGAGTTTTGGTG TCAGCAGTTAGTTTTGGTATGC 2 126-149 (SEQ ID NO: 331)(Nucleotides 19 through 40  of SEQ ID NO: 332) BG281GGTTGGAAACAAAGTCAGAACC ACATCATCAACAGCAAAACCAG 7 195-198 (SEQ ID NO: 333)(Nucleotides 19 through 40  of SEQ ID NO: 334) BG285TGCTTCTTGGTTTCTCATCATC ATGGTTATGTGGGTTGTGTTCA 1 309-316 (SEQ ID NO: 335)(Nucleotides 19 through 40  of SEQ ID NO: 336) BG82TTCCCATATGCAACAGACCTT AACGGTGGTGTGTTTATTGCT 3 195-204 (SEQ ID NO: 337)(Nucleotides 19 through 39  of SEQ ID NO: 338) BG96 TTAACGAGGGTGGTGATGGTTCGATGTTATGGTAGCAGCAA 3 184-191 (SEQ ID NO: 341)(Nucleotides 19 through 39  of SEQ ID NO: 342) BI107AGCAGTGATGTCTTGGCTATGT GTTTCCGGTTCTTTGTCGTTC 5 354-429 (SEQ ID NO: 343)(Nucleotides 19 through 39  of SEQ ID NO: 344) BI113AACATCGTAATGAGGAGGAGGA ACAGTATCAGCAACACCAGCAG 8 241-253 (SEQ ID NO: 345)(Nucleotides 19 through 40  of SEQ ID NO: 346) BI116TCAACCCTTCAGATTTTCTTCC CACACTTTCTCGTTTGCTCTCT 8 218-226 (SEQ ID NO: 347)(Nucleotides 19 through 40  of SEQ ID NO: 348) BI122CAATTTCCTTAGTGGCCGTTAC TTATTAGCTGGGCTTTTCTTCG 7 366-369 (SEQ ID NO: 349)(Nucleotides 19 through 40  of SEQ ID NO: 350) BI68ATCAGCGTAAATTCTGGCCTTA CCATTCCAATCCACACTATCG 5 261-276 (SEQ ID NO: 351)(Nucleotides 19 through 39  of SEQ ID NO: 352) BI75CGTAGGAAGAAGGATCGAGTTC CCCAATTCAAAACGAAGAACC 4 187-193 (SEQ ID NO: 353)(Nucleotides 19 through 39  of SEQ ID NO: 344) BI86CGTCGAAGTCAAAATCAATCTC GAAAAGAAATCACCCCGAAGAT 8 223-249 (SEQ ID NO: 355)(Nucleotides 19 through 40  of SEQ ID NO: 356) BI96CTCATTCACCCAACCAAAATGT GGCTAATTCACCTGTTTCTGCT 4 195-197 (SEQ ID NO: 357)(Nucleotides 19 through 40  of SEQ ID NO: 358) BI98TCAACAGCCAACTCAAAGTGAT CATCAATCAACCCTTTCGTTTC 6 154-164 (SEQ ID NO: 359)(Nucleotides 19 through 40  of SEQ ID NO: 360) MsTri7698CAGTTGATGCATAGAAACGCA AAGCGATTTCATTAGTAGTTGT 8 194-196 (SEQ ID NO: 447)(Nucleotides 19 through 40  of SEQ ID NO: 448) MsTri7729ATCTGGGAAGTGTGACCTCCT TCAAAACCTTGGTGTTGGTTG 4 295-300 (SEQ ID NO: 395)(Nucleotides 19 through 39  of SEQ ID NO: 396) MsTri7771CATACTATGGTGGTGGTTGGG CTCTTTAAGATTGCTTCTCTTGC 8 368-393 (SEQ ID NO: 397)(Nucleotides 19 through 41  of SEQ ID NO: 398) MsTri7807TCACCAGCACATGAATCAAAA AACAACCTAGATTTTCTCGACC 8 238-242 (SEQ ID NO: 449)(Nucleotides 19 through 40  of SEQ ID NO: 450) MsTri8119AGGGTTGATGCAGATGTTACG ATTGCAATCATCTTCTCCCCT 3 270-282 (SEQ ID NO: 451)(Nucleotides 19 through 39  of SEQ ID NO: 452) MsTri8491GGACGGTTTCGAACTTCTAGC CGAGGCATCTTCATCTTCAAC 7 206-222 (SEQ ID NO: 399)(Nucleotides 19 through 39  of SEQ ID NO: 400) MsTri8616AACAATATGATCTGGCATGTCG GGAAGATCACCATTTTGTCCA 7 274-281 (SEQ ID NO: 453)(Nucleotides 19 through 39  of SEQ ID NO: 454) MsTri8637GATAAAGCTCCCACAGTTCCC CTCTTTTCTCTTCAATTTTCAAT 3 232-238 (SEQ ID NO: 401)(Nucleotides 19 through 41  of SEQ ID NO: 402) MsTri8733AGGTACAAGCCATGATGTCCA TTTCCAAACTTTCCTTCTTTTG 6 188-205 (SEQ ID NO: 455)(Nucleotides 19 through 40  of SEQ ID NO: 456) MsTri8791ACAAGAAGAAGATTGCGACGA TGAAGGAAGAAGGAAGAAGGAA 6 178-180 (SEQ ID NO: 457)(Nucleotides 19 through 40  of SEQ ID NO: 458) MsTri8899CGCAGCACATGTAACTTGAAA CACATTCTCTTCGTGCCCTC 8 340-397 (SEQ ID NO: 459)(Nucleotides 19 through 38  of SEQ ID NO: 460) MsTri8923TCCGAAAAAGGTGACAGATTG GGCTCACAACAACAACAAAAT 8 168-192 (SEQ ID NO: 461)(Nucleotides 19 through 39  of SEQ ID NO: 462) MsTri8930CCAAACAGATCTAAAGTTCCCA TGCTTGATTATTGCTAATCGG 3 103-112 (SEQ ID NO: 463)(Nucleotides 19 through 39  of SEQ ID NO: 464) MsTri8931TACAGTTGCCCATACAGGAGG CAAACAGGTGACGAGGTGAAT 3 131-150 (SEQ ID NO: 403)(Nucleotides 19 through 39  of SEQ ID NO: 404) MsTri8949TAAATGCAAGGTAGGTGGTGG CGAGGACGAGTTCTGGTCAA 7 100-145 (SEQ ID NO: 465)(Nucleotides 19 through 38  of SEQ ID NO: 466) MsTri9154AAGACCAAGAGGAATCACCGT TAATTTCATTCGCGATCACAC 1 157-166 (SEQ ID NO: 467)(Nucleotides 19 through 39  of SEQ ID NO: 468) MsTri9223TGAATGTGAGGAAGTGGGTTT CCGCCTCAAATAGTTATAAACTTC 8 140-162(SEQ ID NO: 469) (Nucleotides 19 through 42  of SEQ ID NO: 470)MsTri9326 AGTACTATTGCAATGGCGTGG GGTTTCGCTTGGAATTCTGAT 3 105-107(SEQ ID NO: 471) (Nucleotides 19 through 39  of SEQ ID NO: 472)MsTri9329 ATCAAGATCGACTGAACCACG TTGGCTTTGATTGCTTCAACT 2 117-123(SEQ ID NO: 405) (Nucleotides 19 through 39  of SEQ ID NO: 406)MsTri9475 TGCATGTAATATCTATCTTTGGAA CCAAACCCTAGGAGTCTGAGGT 6 146-146(SEQ ID NO: 568) (SEQ ID NO: 569) MsTri9544 ATTTTTCCACTTCTGGTGGGACAACACAATCATTTTGGGAGC 5 159-177 (SEQ ID NO: 473)(Nucleotides 19 through 39  of SEQ ID NO: 474) MsTri9820TCTTGTTGATATAATCTACGGAA CCTGATGGTCATCACTAAGCC 8 116-120 (SEQ ID NO: 475)(Nucleotides 19 through 39  of SEQ ID NO: 476) MsTri9849TGAGGCTTAACCTTAGGAGGC TTTCAAATCCAAGTGGTGGAG 5 161-167 (SEQ ID NO: 407)(Nucleotides 19 through 39  of SEQ ID NO: 408) MsTri10127GGGAAACCATTTCGTACCCTA AATTCCCAATTCTCATTCGTG 4 123-134 (SEQ ID NO: 409)(Nucleotides 19 through 39  of SEQ ID NO: 410) MsTri10235TTGCCATCGTAGAAAATGGTC CCTTAACACATTTTTGCTTCA 2 353-368 (SEQ ID NO: 411)(Nucleotides 19 through 39  of SEQ ID NO: 412) MsTri10456TGTCGTCTTTTGACCATTTCC TTATCATGTGCAGACAATACC 1 283-296 (SEQ ID NO: 413)(Nucleotides 19 through 39  of SEQ ID NO: 414) MsTri10581CCTTGGCAGCTACAGGTACAG GTCTGCTGCTCCAGCTAAGAA 7 306-316 (SEQ ID NO: 369)(Nucleotides 19 through 39  of SEQ ID NO: 370) MsTri10592GATTAAACATACATGCAACATTGA GGTTGAAATCGACATGAGAGG 8 151-161(SEQ ID NO: 415) (Nucleotides 19 through 39  of SEQ ID NO: 416)MsTri10649 GGATATCCTGGTGGAGGGTAA ACAACCCCATTTCCAACTTTC 1 293-317(SEQ ID NO: 373) (Nucleotides 19 through 39  of SEQ ID NO: 374)MsTri10686 CCAACACTTTAAGCCTCCAAA TGTTCTCCTCTCTTCGTCTCTTG 5 126-132(SEQ ID NO: 417) (Nucleotides 19 through 41  of SEQ ID NO: 418)MsTri10743 CCGGTTCTGTTTGGTAGTGAA AACCAGAGAAAAATCCAACCA 5 111-120(SEQ ID NO: 419) (Nucleotides 19 through 39  of SEQ ID NO: 420)MsTri10801 GGAGCAAACATTCTACCACCA TCACAAAACAAACCCTTCTTCT 5 432-448(SEQ ID NO: 377) (Nucleotides 19 through 40  of SEQ ID NO: 378)MsTri10866 CCTTAGGCACATTGAAAACCA TAAGGGTTCATGCTCACCATC 3 334-340(SEQ ID NO: 421) (Nucleotides 19 through 39  of SEQ ID NO: 422)MsTri11061 AACATGCACAATTAAGCATTCAA ACCTGAAAGGCCACAAAAGAT 5 100-111(SEQ ID NO: 423) (Nucleotides 19 through 39  of SEQ ID NO: 424)MsTri11067 AATTCGGGTGGAATAACAAGC TTGCCTCGGATTATTACTTGTG 3 137-171(SEQ ID NO: 425) (Nucleotides 19 through 40  of SEQ ID NO: 426)MsTri11087 TGACTTAGACACCACCGGAGT TCATCCATTCATTAAAACGCA 3 209-219(SEQ ID NO: 379) (Nucleotides 19 through 39  of SEQ ID NO: 380)MsTri11090 GCAATCACCTTAGCATTTTGG GCCAGTTTTGGGCAATTTTAT 2 187-189(SEQ ID NO: 427) (Nucleotides 19 through 39  of SEQ ID NO: 428)MsTri11131 GTTCAAGCATGGAAAGTTTGG GGGACCTAATATGATGAACTTACA 8 180-188(SEQ ID NO: 429) (Nucleotides 19 through 42  of SEQ ID NO: 430)MsTri11311 TGACAGTTTCCACAATCCTCC GACGAACTCTTTTCTTTTCTGACA 5 305-317(SEQ ID NO: 431) (Nucleotides 19 through 42  of SEQ ID NO: 432)MsTri11314 ATACACCATAGCACGAGACGC TAATTCGAGGAGGATTGTGGA 5 131-137(SEQ ID NO: 381) (Nucleotides 19 through 39  of SEQ ID NO: 382)MsTri11419 ACAAGAAGAAGATTGCGACGA TGAAGGAAGAAGGAAGAAGGAA 6 177-180(SEQ ID NO: 433) (Nucleotides 19 through 40  of SEQ ID NO: 434)MsTri11460 AATTTGGACTTTGATTGTGCG CAAGAACCAGATCATCAACAACA 7 295-306(SEQ ID NO: 435) (Nucleotides 19 through 41  of SEQ ID NO: 436)MsTri11470 GGAGATGAAGAAGGAGATGGG TTGAAATAGTGCAAGAAGAACCC 8 306-319(SEQ ID NO: 385) (Nucleotides 19 through 41  of SEQ ID NO: 386)MsTri11523 TGTCACTTGTTCTGGTCCTTCT GGAGAGAGCAAAGTCTCTTCAA 2 136-142(SEQ ID NO: 387) (Nucleotides 19 through 40  of SEQ ID NO: 388)MsTri11701 AGCTTTTTCAACGAGTTCAGC TTTCATCAACATCAAACACCG 4 173-189(SEQ ID NO: 439) (Nucleotides 19 through 39  of SEQ ID NO: 440)MsTri11744 TTCTTGGCTTCGACTTCTTCA CCGATTGGACTCGGAACTT 2 330-373(SEQ ID NO: 441) (Nucleotides 19 through 37  of SEQ ID NO: 442)MsTri11748 GGATTTCGTTTGGGTTCATTT TCTGTAACACAGGCAGAGTCG 4 310-316(SEQ ID NO: 443) (Nucleotides 19 through 39  of SEQ ID NO: 444)MsTri11989 CAGGAACATAACTGTGACCCG TCCTAATACCCCATTCATTGGT 4 111-112(SEQ ID NO: 389) (Nucleotides 19 through 40  of SEQ ID NO: 390)MsTri12038 GCCTTTAGGCCAATCAGAGAC AAGATTAGGGTTTGAGTAAGGGAA 4 211-216(SEQ ID NO: 391) (Nucleotides 19 through 42  of SEQ ID NO: 392) Mt1D06GAAGGTTTTGGGTGGTGATG CCATGGCTCTTTCCTACCAA 7 167-189 (SEQ ID NO: 479)(Nucleotides 19 through 38  of SEQ ID NO: 480) Mt1G03TGGTTGATCAATGTTCCTCCT AAAGAGATTGGGTCGGTGAA 8 238-269 (SEQ ID NO: 481)(Nucleotides 19 through 38  of SEQ ID NO: 482) MtBA36F01F1AATAAACACAGATTCCAAATCCA TCTTCATCGCTTTCTTCTATTTCA 1 126-145(SEQ ID NO: 483) (Nucleotides 19 through 42  of SEQ ID NO: 484)MtBC01G06F3 TCAGGACAAACTGCCATTTC TGCATTGAAGCAAATTAACGA 1 177-189(SEQ ID NO: 485) (Nucleotides 19 through 39  of SEQ ID NO: 486) MTIC107TACGTAGCCCCTTGCTCATT CAAACCATTTCCTCCATTGTG 1 170-184 (SEQ ID NO: 487)(Nucleotides 19 through 39  of SEQ ID NO: 488) MTIC124TTGGGTTGTCAATAATGCTCA TTGTCACGAGTGTTGGAATTTT 3 135-192 (SEQ ID NO: 489)(Nucleotides 19 through 40  of SEQ ID NO: 490) MTIC169GCGTGCTAGGTTTGAGAGGA TCAAAACCCTAAAACCCTTTCTC 3  99-113 (SEQ ID NO: 491)(Nucleotides 19 through 41  of SEQ ID NO: 492) MTIC19TGCAACAGAAGAAGCAAAACA TCTAGAAAAAGCAATGATGTGAGA 2 149-166(SEQ ID NO: 495) (Nucleotides 19 through 42  of SEQ ID NO: 496) MTIC233AAGGAACAATCCCAGTTTTT GCGTAACGTAACAACATTCA 1 145-170 (SEQ ID NO: 497)(Nucleotides 19 through 38  of SEQ ID NO: 498) MTIC238CCTTAGCCAAGCAAGTAAAA TTCTTCTTCTAGGAATTTGGAG 5 140-144 (SEQ ID NO: 499)(Nucleotides 19 through 40  of SEQ ID NO: 500) MTIC247TGAGAGCATTGATTTTTGTG TTCGCAGAACCTAAATTCAT 1 125-131 (SEQ ID NO: 501)(Nucleotides 19 through 38  of SEQ ID NO: 502) MTIC248GGATTGTGATGAAGAAATGG TATCTCCCTTCTCCTTCTCC 8 137-154 (SEQ ID NO: 503)(Nucleotides 19 through 38  of SEQ ID NO: 504) MTIC249GTGGGTGAGGATGTGTGTAT TAGGTCATGGCTATTGCTTC 4 122-131 (SEQ ID NO: 505)(Nucleotides 19 through 38  of SEQ ID NO: 506) MTIC250CGTTGATGATGTTCTTGATG GCCTGAACTATTGTGAATGG 6 133-136 (SEQ ID NO: 507)(Nucleotides 19 through 38  of SEQ ID NO: 508) MTIC258TGAAATTCACATCAACTGGA CACCACCTTCACCTAAGAAA 1 147-151 (SEQ ID NO: 509)(Nucleotides 19 through 38  of SEQ ID NO: 510) MTIC304AGCGTAAAGTAAAACCCTTTC TTGGGCTTAATTTGACTGAT 2 159-175 (SEQ ID NO: 511)(Nucleotides 19 through 38  of SEQ ID NO: 512) MTIC332GGTCATACGAGCTCCTCCAT CCCTGGGTTTTTGATCCAG 4 148-163 (SEQ ID NO: 513)(Nucleotides 19 through 37  of SEQ ID NO: 514) MTIC338CATTGGTGGACGAGGTCTCT TCCCCTTAAGCTTCACTCTTTTC 3 181-196 (SEQ ID NO: 515)(Nucleotides 19 through 41  of SEQ ID NO: 516) MTIC343CCATTGCGGTGGCTACTCT TCCGATCTTGCGTCCTAACT 6 140-160 (SEQ ID NO: 517)(Nucleotides 19 through 38  of SEQ ID NO: 518) MTIC35GGCAGGAACAGATCCTTGAA GAAGAAGAAAAAGAGATAGATCTGTGG 7 129-132(SEQ ID NO: 519) (Nucleotides 19 through 45  of SEQ ID NO: 520) MTIC354AACCTACGCTAGGGTTGCAG AAGTGCCAAAGAACAGGGTTT 2 244-257 (SEQ ID NO: 521)(Nucleotides 19 through 39  of SEQ ID NO: 522) MTIC452TCACAAAAACTGCATAAAGC CTAGTGCCAACACAAAAACA 2 114-126 (SEQ ID NO: 523)(Nucleotides 19 through 38  of SEQ ID NO: 524) MTIC51ACAAAAACTCTCCCGGCTTT AGTATAGTGATGAAGTGGTAGTGAACA 3 141-154(SEQ ID NO: 527) (Nucleotides 19 through 45  of SEQ ID NO: 528) MTIC82GAGAGGATTTCGGTGATGT CACTTTCCACACTCAAACCA 7 138-142 (SEQ ID NO: 570)(SEQ ID NO: 571)  MTIC84 GGGAAAAGGTGTAGCCATTG TCTGAGAGAGAGACAAACAAAACAA1 183-193 (SEQ ID NO: 529) (Nucleotides 19 through 43 of SEQ ID NO: 530) MTIC95 AGGAAGGAGAGGGACGAAAG AAAGGTGTTGGGTTTTGTGG 1146-148 (SEQ ID NO: 533) (Nucleotides 19 through 38  of SEQ ID NO: 534)RCS0121 CTGCTTTGGTTTGGAAGAAA GGAAAGAATATGCAATTTCTCGAT 2  92-100(SEQ ID NO: 535) (Nucleotides 19 through 42  of SEQ ID NO: 536) RCS1209TGAACTTTGAAGCCACATTGA AAAATCCAGAAGCACGAGTGA 4 109-111 (SEQ ID NO: 537)(Nucleotides 19 through 39  of SEQ ID NO: 538) RCS2510GCCCTAAAAGTTGAAAGAGCA CACGAGGGAACACTTCATCA 6 122-220 (SEQ ID NO: 539)(Nucleotides 19 through 38  of SEQ ID NO: 540) RCS2936CCAATGCAATTCGGTAATCC CGTTATTTATCCCTCCGGGT 8 141-376 (SEQ ID NO: 541)(Nucleotides 19 through 38  of SEQ ID NO: 542) RCS4209TCACAATGGGCACCTAATCA CAATTTTCGCTGACTGACCA 2 157-158 (SEQ ID NO: 543)(Nucleotides 19 through 38  of SEQ ID NO: 544) RCS4310GCCATTTGCTTCAACCTTGT GCCATTGCTGGAATCGTAAT 4 269-272 (SEQ ID NO: 545)(Nucleotides 19 through 38  of SEQ ID NO: 546) TC105099AGATAGGAATTTGGGTCGGG ACAACCATGATGTGGGAATG 5 111-117 (SEQ ID NO: 553)(Nucleotides 19 through 38  of SEQ ID NO: 554) TC106861GCAGGGCTGAGACTCCAGTA AGCCCTGCTTTTTCTCCTCT 5 245-247 (SEQ ID NO: 555)(Nucleotides 19 through 38  of SEQ ID NO: 556) TC85780-1AAAGTGACATGATCCACAGG GCTAAGAAAGCATGGGGTTGTTGG 5 276-283 (SEQ ID NO: 557)(Nucleotides 19 through 42  of SEQ ID NO: 558)

TABLE 6 Number of simplex, duplex, double simplex and co-dominant SSRmarkers used to construct tetraploid linkage maps in each of theparental alfalfa genotypes. Altet-4 NECS-141 LG 1:1 5:1 3:1 Co-dominant1:1 5:1 3:1 Co-dominant 1 20 12 4 7 23 9 4 5 2 23 14 3 9 26 2 3 6 3 19 57 8 41 8 7 13 4 38 8 5 17 24 3 5 8 5 33 3 6 10 22 7 6 7 6 29 2 1 11 19 21 4 7 22 2 0 5 28 11 0 8 8 14 13 0 3 48 6 0 13 Total 198 59 26 70 231 4826 64

Example 10 Further QTL Analysis Using SF-ANOVA from Callus and WholePlant Assays

Twenty markers associated with the response to Al in the callus bioassaywere identified using SF-ANOVA (Table 7). Of these markers, 14 wereassociated with decreasing total callus weight ratio (TCWR) and six withincreasing TCWR. Forty-one markers relevant to Al tolerance in the wholeplant assay were also found using SF-ANOVA. Of these, 21 markers wereassociated with increasing total root length ratio (TRLR) and 20 markerswere associated with decreasing TRLR.

Using interval mapping, a QTL for callus growth was identified at 90 cMon LG 1 from Altet-4 (FIG. 9A). This QTL explained 20.8% of thephenotypic variation for TCWR. The average TCWR score of the alleliccombination Q₁₂ (0.97) was higher than the other possible alleliccombinations at this locus, which had an average TCWR score of 0.75(FIG. 9B). All allelic combinations were represented by at least 16individuals. These results suggest that a recessive allele providingincreased Al-tolerance is present on homologues 1 and 2 in a duplexcondition. The SF-ANOVA did not identify a marker with a positiveassociation for TCWR in LG 1 of Altet-4, likely due to the lack of aduplex marker associated with Q₁ and Q₂ in the QTL region. However, twosimplex markers on homolog H3 in the region of the QTL decreased TCWR(Table 7).

Two QTLs for Al tolerance were identified based on interval mapping ofthe root growth differences in the whole plant assay on LG 4 of Altet-4(FIG. 10) and LG 7 of NECS-14 (FIG. 11). These QTL explained 15.2% and21.7% of the variation, respectively, and again suggested the presenceof recessive alleles that improve Al tolerance. For the Al tolerance QTLfrom Altet-4 located on LG 4 (FIG. 10B), the average TRLR of alleliccombination Q₃₄ (0.72) was higher than the average TRLR from all otherallelic combinations (0.52). The results from the SF-ANOVA show thatamong the Altet-4 markers on LG 4, five simplex markers on homologs H3and H4 were positively associated with Al tolerance, while one simplexmarker on homolog H1 and one duplex marker bridging homolog H1 and H2were negatively associated with Al tolerance (Table 7).

Interval mapping was used to identify additional QTLs for Al toleranceon LGs 4 and 7 (FIGS. 10-11) from evaluations at the whole plant level.Soil-based evaluations of the Altet-4×NECS-141 population identified aQTL for root dry weight ratio that represents the relative root growthin unlimed vs limed soil conditions associated with the same markers onLG 4 (data not shown). These represent novel Al tolerance QTLs notpreviously identified in diploids using the callus bioassay. These QTLare apparently relevant at the whole plant level but not in callus. Thelack of correlation between Al tolerance responses in the callusbioassay and whole plant assay suggests that although similar stressresponses may be involved, these systems capture different tolerancemechanisms. Al tolerance evaluations at the whole plant level may thuscapture defense mechanisms at the cell level as well as complex organresponses, including changes in root growth. The primary effects ofgrowth inhibition due to Al⁺³ occur at or near the root tip (Kochian etal. Ann Rev. Pl. Biol. 55:459-493, 2004). Alternatively, the additionalAl tolerance QTL may have been identified due to the increase in markerdensity compared to the relatively sparse genetic map used in a diploidmapping study (Narasimhamoorthy et al. TAG 114:901-909, 2007), or theyrepresent QTL that are only relevant at the tetraploid level due toallelic interactions or gene expression changes. Additionally, thetetraploid and diploid populations used to identify Al tolerance QTLdiffer in their genetic background. The identification of QTLs may varyon the genetic background of the populations used (Monteros et al. CropSci. 48:2223-2234, 2008; Tang and Scarth Pl. Breeding 123:254-261,2004). In Oryza sativa L., the effect of genetic background on QTLsidentified was greater than the environmental effects (Liao et al. TAG103:104-111, 2001).

Al tolerance QTL on LG 7 of NECS-141 explained 21.7% of the phenotypicvariation for total root length ratio (TRLR) from the whole plant assayin media. For the Al tolerance QTL identified from the whole plant assayon LG 7 from NECS-141, the average TRLR of the allelic combination Q₂₃(0.75), was higher than the average TRLR of all other alleliccombinations (0.50) (FIG. 11B). No marker significantly associated withthe Al tolerance phenotype on LG 7 was identified in the SF-ANOVA,likely due to the absence of markers covering homologs H2 and H3 ofNECS-141 in this region (e.g. FIG. 8). However, four simplex markers onhomologous chromosome H1 were negatively associated with TRLR thusproviding additional evidence showing that the recessive allele on H2and H3 increases Al tolerance at the whole plant level (Table 7).

TABLE 7 Additional significant markers associated with Al tolerance inalfalfa from the callus bioassay (CBA) and whole plant assay in media(WPA-M) based on single-factor ANOVA (p < 0.05). LG^(a) H^(b) cM ParentMarker Effect^(c) Mean(0)^(d) Mean(1)^(e) SED^(f) p value Callusbioassay LG1 H3 75.7 Altet-4 MTIC233-149A − 0.817 0.745 0.035 0.041 H357.1 Altet-4 MtBA36F01F1-126A − 0.828 0.740 0.034 0.011 LG3 H12 50.1NECS-141 1c09gat6-1-211 − 0.878 0.772 0.047 0.025 H12 59.8 NECS-141MsTri9326-107 − 0.863 0.771 0.044 0.039 LG4 H1 41.1 Altet-41h09aat11-1-237 − 0.825 0.752 0.035 0.041 H14 55.2 Altet-4MsTri11701-176 − 0.908 0.774 0.054 0.015 H2 25.3 NECS-1411h09aat11-1-233 + 0.756 0.831 0.036 0.037 LG5 H4 0 Altet-42c06gat6-1-128A − 0.847 0.751 0.034 0.006 H4 21.3 Altet-4 BG157-154 −0.829 0.737 0.034 0.008 H4 88.1 NECS-141 2c06gat6-1-137 + 0.754 0.8270.034 0.036 LG6 H2 67.9 NECS-141 3d03atc5-1-244 + 0.752 0.829 0.0340.025 H2 77.5 NECS-141 MTIC250-133 + 0.751 0.823 0.034 0.039 H1 72.1NECS-141 MTIC343-140 − 0.823 0.737 0.034 0.015 H2 72.1 NECS-141MTIC343-143 + 0.749 0.838 0.034 0.009 LG7 H1 56.2 Altet-41b11gtg6-1-313A + 0.762 0.832 0.035 0.048 H4 8.4 Altet-4 BF26-296A −0.824 0.752 0.034 0.036 H4 6.5 Altet-4 BF56-296A − 0.824 0.752 0.0340.036 H2 53.4 Altet-4 AW212-265 − 0.829 0.732 0.035 0.007 H1 1.0NECS-141 BF26-306 − 0.839 0.740 0.033 0.004 H1 0.1 NECS-141 BF56-306 −0.837 0.743 0.033 0.006 Whole plant assay in media LG1 H24 68.6 NECS-141BG137-323 − 0.582 0.481 0.041 0.013 H1 90.7 NECS-141 BG248-348 + 0.4690.527 0.029 0.046 LG2 H34 90.9 Altet-4 BF111-173A − 0.629 0.530 0.0440.025 H4 10.9 NECS-141 AW16-234 − 0.580 0.510 0.035 0.046 H4 8.2NECS-141 MTIC19-160 − 0.585 0.507 0.035 0.026 LG3 H3 8.1 Altet-4BE41-223 + 0.469 0.528 0.029 0.044 H3 5.4 Altet-4 BF220-299 + 0.4680.531 0.029 0.031 H3 8.5 Altet-4 BF225-201 + 0.469 0.528 0.029 0.044 H46.2 Altet-4 BG115-227A − 0.527 0.459 0.029 0.021 H3 5.0 Altet-4MsTri8931-131 + 0.470 0.531 0.029 0.038 H14 16.6 Altet-4 BG272-456 −0.582 0.469 0.036 0.002 H1 55.5 NECS-141 BF120-224 + 0.469 0.530 0.0290.039 H3 61.6 NECS-141 MtBA36F01F1-140 − 0.522 0.458 0.030 0.032 LG4 H367.5 Altet-4 1g05tct12-1-268A + 0.463 0.537 0.029 0.013 H3 73.1 Altet-4AW232-195 + 0.463 0.535 0.029 0.014 H1 26.3 Altet-4 AW260-254 − 0.5320.456 0.029 0.009 H4 24.8 Altet-4 BE84-229A + 0.462 0.530 0.029 0.022H12 27.3 Altet-4 BG166-132A − 0.589 0.478 0.039 0.005 H4 31.5 Altet-4MsTri9857-193A + 0.468 0.526 0.029 0.049 H3 48.7 Altet-4 RCS1209-109A +0.467 0.538 0.029 0.016 LG5 H14 16.2 Altet-4 MsTri11314-131 − 0.5470.479 0.034 0.048 H4 54.6 NECS-141 2c12tta5-1-316 − 0.525 0.456 0.0290.021 H3 62.3 NECS-141 AW369-169 + 0.451 0.536 0.029 0.004 H12 96.7NECS-141 AW389-486 − 0.569 0.480 0.039 0.025 H3 96.7 NECS-141AW389-489 + 0.460 0.526 0.029 0.026 H3 95.7 NECS-141 MsTri10801-447 +0.454 0.532 0.029 0.007 LG6 H1 0 Altet-4 1f11aatt4-1-192A − 0.533 0.4710.030 0.037 H3 51.2 Altet-4 3d03cat7-1-303A + 0.441 0.516 0.033 0.024H23 28.4 Altet-4 BF149-107A + 0.431 0.511 0.037 0.033 H14 66.0 Altet-4MTIC250-136A − 0.544 0.476 0.032 0.034 H3 72.3 Altet-4 MTIC343-160 +0.410 0.509 0.043 0.023 H1 0 NECS-141 1c06tta6-1-214 − 0.525 0.463 0.0290.034 H2 67.9 NECS-141 3d03atc5-1-244 + 0.466 0.527 0.029 0.038 H1 26.4NECS-141 BI98-164 − 0.523 0.463 0.029 0.045 LG7 H1 57.4 NECS-141BF142-266 − 0.528 0.459 0.029 0.019 H1 1.0 NECS-141 BF26-306 − 0.5330.459 0.029 0.011 H1 0.1 NECS-141 BF56-306 − 0.529 0.462 0.029 0.023 LG8H2 87.3 Altet-4 BI86-223 + 0.460 0.522 0.030 0.038 H12 21.3 NECS-141AW186-237 + 0.379 0.500 0.058 0.042 H12 20.3 NECS-141 AW255-234 + 0.3760.503 0.062 0.046 H13 75.2 NECS-141 MsTri11470-319 − 0.579 0.463 0.0630.069 ^(a)Linkage group ^(b)Homologous chromosome number ^(c)Effect: (+)presence of the marker increases the trait value; (−) absence of themarker increases the trait value ^(d)Mean of individuals with markergenotype 0 (absent) ^(e)Mean of individuals with marker genotype 1(present) ^(f)standard error of the difference between marker classmeans

What is claimed is:
 1. A method for increasing the aluminum tolerance ofan alfalfa line, said method comprising introgressing at least onechromosomal locus contributing to aluminum tolerance from a parentalfalfa plant into a selected alfalfa line, wherein said chromosomallocus maps between loci Mstri9857-18793A97 and AW260-24554 on linkagegroup
 4. 2. The method of claim 1, wherein the aluminum tolerant alfalfaplant is an agronomically elite plant.
 3. The method of claim 1, whereinthe aluminum tolerant alfalfa plant is a hybrid or inbred plant.
 4. Themethod of claim 1, wherein the introgressing is by marker-assistedselection using at least a first genetic marker linked to saidchromosomal locus.
 5. The method of claim 4, wherein the marker isselected from one of those detectable using a primer pair in Table
 1. 6.The method of claim 1, wherein the parent alfalfa plant is Altet-4. 7.The method of claim 1, wherein the parent alfalfa plant is a Medicagosativa NECS-141 plant.
 8. The method of claim 1, wherein the parentalfalfa plant exhibits at least a 50% reduction in aluminum sensitivityrelative to the less aluminum tolerant alfalfa line.
 9. The method ofclaim 8, wherein the parent alfalfa plant displays at least a 75%reduction in aluminum sensitivity relative to the less aluminum tolerantalfalfa line.
 10. The method of claim 1 further comprising producing analfalfa seed by crossing the aluminum tolerant plant with itself or asecond alfalfa plant and allowing seed to form.
 11. A method forobtaining an alfalfa plant comprising an allele conferring aluminumtolerance, said method comprising: a) obtaining nucleic acids from analfalfa plant comprising at least a first allele that confers aluminumtolerance, wherein said allele maps between loci Mstri9857-18793A97 andAW260-24554 on linkage group 4; b) assaying said nucleic acids for thepresence of at least a first genetic marker that is genetically linkedto said allele; and c) selecting the alfalfa plant based on the presenceof said genetic marker.
 12. The method of claim 11, wherein the alfalfaplant is a progeny of a plant of Altet-4.
 13. The method of claim 11,wherein the alfalfa plant is a progeny of a plant of a Medicago sativaNECS-141 plant.